These authors contributed equally to this work.

#### **References**

**6. Future perspectives and challenges**

72 Advances in Molecular Retrovirology

A reservoir of latent HIV is the main obstacle in finding a functional and sterilizing cure. Several challenges need to be addressed in order to overcome this obstacle. Defining the latent reservoir is impeded by the rare occurrence of a latent infection in a high background of defective proviral integration. Although HIV prefers integration in or near transcriptionally active genes which leaves ample room for variation in chromatin environment and available host transcription factors. This puts considerable demands on LRAs. LRAs should be effective, yet specific, without being toxic. As LRAs act via pathways involved in distinct cellular processes, pleiotropic effects are to be expected. Furthermore, recent studies on material obtained from HIV-1-positive suppressed patients revealed that currently available LRAs are not strong enough to reactivate the whole pool of latent proviruses, even after multiple rounds of stimulation. One of the concerns arising from "shock and kill" therapy is whether putative LRAs are strong enough to drive virus production to a level at which the immune system will be able to recognize and destroy HIV-1-producing cells. Indeed, trials aiming at testing HDAC inhibitors are inconsistent in showing depletion of latently infected cells while showing increased proviral transcription [407–412]. A complementary strategy would be to use multiple LRAs in combination to broadly and potentially synergistically reactivate the diversely integrated latent proviruses. Synergism between LRAs was already identified, e.g., Vorinostat and Prostratin [84]. Therefore, the quest for identification and characterization of novel compounds which are able to reactivate HIV-1 transcription as well as identifying combina‐ tions of drugs that can synergize to reverse latency is needed. Currently, no cell model is able to recapitulate the complexities of latency *in vivo*. A better system that more closely resembles the *in vivo* situation would greatly aid the understanding of molecular mechanisms underlying latency and the screening of new LRA. Moreover, as HIV-1 persists in a silent state, it contrib‐ utes to a low level of inflammation, which over time leads to immune exhaustion. Furthermore, depletion of cells harboring latent provirus requires antigen-specific CTLs stimulation [399]. Most likely successful eradication therapies will be based on the combination of LRAs coupled with boosting HIV-1-specific immune response. A "shock and kill" approach in combination

with immune therapies provides hope for reversing HIV-1 infection.

, Tokameh Mahmoudi\*

Department of Biochemistry, Erasmus Medical Centre, Rottrerdam, The Netherlands

, Mateusz Stoszko#

These authors contributed equally to this work.

\*Address all correspondence to: t.mahmoudi@erasmusmc.nl

**Author details**

Michael D. Röling#

#


[28] Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio F a, Yassine-Diab B, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic pro‐ liferation. Nat. Med. 2009;15:893–900.

[14] Pan X, Baldauf H-M, Keppler OT, Fackler OT. Restrictions to HIV-1 replication in resting CD4+ T lymphocytes. Cell Res. Shanghai Institutes for Biological Sciences,

[15] Donahue D a, Wainberg M a. Cellular and molecular mechanisms involved in the es‐

[16] Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc.

[17] Stevenson M, Stanwick TL, Dempsey MP, Lamonica CA. HIV-1 replication is control‐ led at the level of T cell activation and proviral integration. EMBO J. 1990;9:1551–60.

[18] Wang W, Guo J, Yu D, Vorster PJ, Chen W, Wu Y. A dichotomy in cortical actin and chemotactic actin activity between human memory and naive T cells contributes to their differential susceptibility to HIV-1 infection. J. Biol. Chem. 2012;287:35455–69.

[19] Spear M, Guo J, Wu Y. The trinity of the cortical actin in the initiation of HIV-1 infec‐

[20] Siliciano RF, Greene WC. HIV latency. Cold Spring Harb. Perspect. Med.

[21] Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature.

[22] Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, et al. Viral dynam‐ ics in human immunodeficiency virus type 1 infection. Nature. 1995;373:117–22.

[23] Chavez L, Calvanese V, Verdin E. HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells. PLoS Pathog. 2015;11:e1004955.

[24] Eriksson S, Graf EH, Dahl V, Strain MC, Yukl SA, Lysenko ES, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog.

[25] Chun T, Carruth L, Finzi D, Shen X, DiGiuseppe J, Taylor H, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Lett. to Nat.

[26] Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1,

[27] Strain MC, Günthard HF, Havlir D V, Ignacio CC, Smith DM, Leigh-Brown AJ, et al. Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence. Proc. Natl. Acad. Sci. U. S. A. 2003;100:4819–24.

even in patients on effective combination therapy. Nat. Med. 1999;5:512–7.

tablishment of HIV-1 latency. Retrovirology. Retrovirology; 2013;10:11.

Chinese Academy of Sciences; 2013;23:876–85.

Natl. Acad. Sci. U. S. A. 1997;94:1925–30.

tion. Retrovirology. 2012;9:45.

Public Library of Science; 2013;9:e1003174.

2011;1:a007096.

74 Advances in Molecular Retrovirology

1995;373:123–6.

1997;246:170–170.


[53] Henrich TJ, Hu Z, Li JZ, Sciaranghella G, Busch MP, Keating SM, et al. Long-term re‐ duction in peripheral blood HIV type 1 reservoirs following reduced-intensity condi‐ tioning allogeneic stem cell transplantation. J. Infect. Dis. 2013;207:1694–702.

[41] Chun T-W, Nickle DC, Justement JS, Meyers JH, Roby G, Hallahan CW, et al. Persis‐ tence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral ther‐

[42] Lerner P, Guadalupe M, Donovan R, Hung J, Flamm J, Prindiville T, et al. The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy. J. Vi‐

[43] Yilmaz A, Verhofstede C, D'Avolio A, Watson V, Hagberg L, Fuchs D, et al. Treat‐ ment intensification has no effect on the HIV-1 central nervous system infection in patients on suppressive antiretroviral therapy. J. Acquir. Immune Defic. Syndr.

[44] Symons J, Vandekerckhove L, Hütter G, Wensing AMJ, Van Ham PM, Deeks SG, et al. Dependence on the CCR5 coreceptor for viral replication explains the lack of re‐ bound of CXCR4-predicted HIV variants in the Berlin patient. Clin. Infect. Dis.

[45] Hütter G, Nowak D, Mossner M, Ganepola S, Müssig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N. Engl. J. Med.

[46] Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a

[47] Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature.

[48] Libert F, Cochaux P, Beckman G, Samson M, Aksenova M, Cao A, et al. The deltaccr5 mutation conferring protection against HIV-1 in Caucasian populations has a single

[49] Yukl SA, Boritz E, Busch M, Bentsen C, Chun T-W, Douek D, et al. Challenges in de‐ tecting HIV persistence during potentially curative interventions: a study of the Ber‐

[50] Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the

[51] Sabeti PC, Walsh E, Schaffner SF, Varilly P, Fry B, Hutcheson HB, et al. The case for selection at CCR5-Delta32. PLoS Biol. Public Library of Science; 2005;3:e378.

[52] Yukl SA, Boritz E, Busch M, Bentsen C, Chun T-W, Douek D, et al. Challenges in de‐ tecting HIV persistence during potentially curative interventions: a study of the Ber‐

and recent origin in Northeastern Europe. Hum. Mol. Genet. 1998;7:399–406.

lin patient. PLoS Pathog. Public Library of Science; 2013;9:e1003347.

lin patient. PLoS Pathog. Public Library of Science; 2013;9:e1003347.

CCR5 gene 32-basepair deletion. Nat. Genet. 1997;16:100–3.

major co-receptor for primary isolates of HIV-1. Nature. 1996;381:661–6.

apy. J. Infect. Dis. 2008;197:714–20.

rol. 2011;85:4772–82.

2010;55:590–6.

76 Advances in Molecular Retrovirology

2014;59:596–600.

2009;360:692–8.

1996;381:667–73.


[80] Weiss A, Wiskocil RL, Stobo JD. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occur‐ ring at a pre-translational level. J. Immunol. 1984;133:123–8.

[66] Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, et al. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV. N. Engl. J. Med. 2014;370:901–

[67] Allers K, Schneider T. CCR5Δ32 mutation and HIV infection: basis for curative HIV

[68] Vrins CLJ, Out R, van Santbrink P, van der Zee A, Mahmoudi T, Groenendijk M, et al. Znf202 affects high density lipoprotein cholesterol levels and promotes hepatos‐

[69] Huelsmann PM, Hofmann AD, Knoepfel SA, Popp J, Rauch P, Di Giallonardo F, et al. A suicide gene approach using the human pro-apoptotic protein tBid inhibits HIV-1

[70] Hamer DH. Can HIV be Cured? Mechanisms of HIV persistence and strategies to

[71] Deeks SG, Autran B, Berkhout B, Benkirane M, Cairns S, Chomont N, et al. Towards an HIV cure: a global scientific strategy. Nat. Rev. Immunol. Nature Publishing

[72] Barouch DH, Deeks SG. immunologic strategies for HIV-1 remission and eradication.

[73] Archin NM, Margolis DM. Emerging strategies to deplete the HIV reservoir. Curr.

[74] Folks TM, Clouse KA, Justement J, Rabson A, Duh E, Kehrl JH, et al. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically

[75] Folks TM, Justement J, Kinter a, Dinarello C a, Fauci a S. Cytokine-induced expres‐ sion of HIV-1 in a chronically infected promonocyte cell line. Science. 1987;238:800–2.

[76] Emiliani S, Van Lint C, Fischle W, Paras P, Ott M, Brady J, et al. A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency. Proc.

[77] Emiliani S, Fischle W, Ott M, Van Lint C, Amella CA, Verdin E. Mutations in the tat Gene Are Responsible for Human Immunodeficiency Virus Type 1 Postintegration

[78] Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a latent infection after

[79] Folks TM, Justement J, Kinter A, Dinarello CA, Fauci AS. Cytokine-induced expres‐ sion of HIV-1 in a chronically infected promonocyte cell line. Science. 1987;238:800–2.

infected T-cell clone. Proc. Natl. Acad. Sci. U. S. A. 1989;86:2365–8.

10.

78 Advances in Molecular Retrovirology

therapy. Curr. Opin. Virol. 2015;14:24–9.

replication. BMC Biotechnol. 2011;11:4.

combat it. Curr. HIV Res. 2004;2:99–111.

Group; 2012;12:607–14.

Science (80-.). 2014;345:169–74.

Opin. Infect. Dis. 2014;27:29–35.

Natl. Acad. Sci. U. S. A. 1996;93:6377–81.

Latency in the U1 Cell Line. J. Virol. 1998;72:1666–70.

acute infection of T cells in vitro. EMBO. 2003;22:1868–77.

teatosis in hyperlipidemic mice. PLoS One. 2013;8:e57492.


[106] Goldstone DC, Ennis-Adeniran V, Hedden JJ, Groom HCT, Rice GI, Christodoulou E, et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphos‐ phohydrolase. Nature. Nature Publishing Group; 2011;480:379–82.

[93] De Crignis E, Mahmoudi T. HIV eradication: combinatorial approaches to activate la‐

[94] Archin NM, Sung JM, Garrido C, Soriano-Sarabia N, Margolis DM. Eradicating HIV-1 infection: seeking to clear a persistent pathogen. Nat. Rev. Microbiol. Nature

[95] Denton PW, Olesen R, Choudhary SK, Archin NM, Wahl a., Swanson MD, et al. Gen‐

[96] Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno F a, et al. Humanized mice mount specific adaptive and innate immune responses to EBV and

[97] Denton PW, Estes JD, Sun Z, Othieno F a., Wei BL, Wege AK, et al. Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT

[98] North TW, Higgins J, Deere JD, Hayes TL, Villalobos A, Adamson L, et al. Viral sanc‐ tuaries during highly active antiretroviral therapy in a nonhuman primate model for

[99] Van Rompay KK a. Evaluation of antiretrovirals in animal models of HIV infection.

[100] Hatziioannou T, Ambrose Z, Chung NPY, Piatak M, Yuan F, Trubey CM, et al. A macaque model of HIV-1 infection. Proc. Natl. Acad. Sci. U. S. A. 2009;106:4425–9.

[101] Krebs FC, Hogan TH, Quiterio S, Gartner S, Wigdahl B. Lentiviral LTR-directed Ex‐ pression, Sequence Variation, and Disease Pathogenesis Reviews. In: CL K, B F, B H, B K, F M, PA M, et al., editors. HIV Seq. Compend. 2001. Los Alamos: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory: Los Alamos, NM;

[102] Bruner KM, Hosmane NN, Siliciano RF. Towards an HIV-1 cure: measuring the la‐

[103] Hindson BJ, Ness KD, Masquelier D a., Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA

[104] Procopio FA, Fromentin R, Kulpa DA, Brehm JH, Bebin A-G, Strain MC, et al. A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-in‐

[105] Descours B, Cribier A, Chable-Bessia C, Ayinde D, Rice G, Crow Y, et al. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells. Retrovirology. Retro‐

tent reservoir. Trends Microbiol. Elsevier Ltd; 2015;23:192–203.

copy number. Anal. Chem. 2011;83:8604–10.

fected Individuals. EBioMedicine. Elsevier; 2015;

eration of HIV Latency in Humanized BLT Mice. J. Virol. 2012;86:630–4.

tent viruses. Viruses. 2014;6:4581–608.

80 Advances in Molecular Retrovirology

Publishing Group; 2014;12:750–64.

TSST-1. Nat. Med. 2006;12:1316–22.

mice. PLoS Med. 2008;5:0079–89.

AIDS. J. Virol. 2010;84:2913–22.

Antiviral Res. 2010;85:159–75.

2001. p. 1–42.

virology; 2012;9:87.


[131] Brady T, Agosto LM, Malani N, Berry CC, O'Doherty U, Bushman F. HIV integration site distributions in resting and activated CD4+ T cells infected in culture. AIDS. 2009;23:1461–71.

[119] Maroun M, Delelis O, Coadou G, Bader T, Ségéral E, Mbemba G, et al. Inhibition of early steps of HIV-1 replication by SNF5/Ini1. J. Biol. Chem. 2006;281:22736–43. [120] Turelli P, Doucas V, Craig E, Mangeat B, Klages N, Evans R, et al. Cytoplasmic re‐ cruitment of INI1 and PML on incoming HIV preintegration complexes: Interference

[121] Ocwieja KE, Brady TL, Ronen K, Huegel A, Roth SL, Schaller T, et al. HIV integration targeting: A pathway involving transportin-3 and the nuclear pore protein RanBP2.

[122] Pierson TC, Kieffer TL, Ruff CT, Buck C, Gange SJ, Siliciano RF. Intrinsic Stability of Episomal Circles Formed during Human Immunodeficiency Virus Type 1 Replica‐

[123] Pierson TC, Zhou Y, Kieffer TL, Christian T, Buck C, Siliciano RF, et al. Molecular Characterization of Preintegration Latency in Human Immunodeficiency Virus Type

[124] Zamborlini A, Lehmann-Che J, Clave E, Giron M-L, Tobaly-Tapiero J, Roingeard P, et al. Centrosomal pre-integration latency of HIV-1 in quiescent cells. Retrovirology.

[125] Trinité B, Ohlson EC, Voznesensky I, Rana SP, Chan CN, Mahajan S, et al. An HIV-1 replication pathway utilizing reverse transcription products that fail to integrate. J.

[126] Han Y, Lassen K, Monie D, Ahmad R, Shimoji S, Liu X, et al. Resting CD4 + T Cells from Human Immunodeficiency Virus Type 1 (HIV-1) -Infected Individuals Carry Integrated HIV-1 Genomes within Actively Transcribed Host Genes. J. Virol.

[127] Liu H, Dow EC, Arora R, Kimata JT, Bull LM, Arduino RC, et al. Integration of hu‐ man immunodeficiency virus type 1 in untreated infection occurs preferentially

[128] Lewinski MK, Bisgrove D, Shinn P, Chen H, Hoffmann C, Hannenhalli S, et al. Ge‐ nome-Wide Analysis of Chromosomal Features Repressing Human Immunodeficien‐

[129] Marini B, Kertesz-farkas A, Ali H, Lucic B, Lisek K, Manganaro L, et al. Nuclear ar‐ chitecture dictates HIV-1 integration site selection. Nature. 2015;521(7551):227-31 [130] Wang GP, Ciuffi A, Leipzig J, Berry CC, Bushman FD. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic

with early steps of viral replication. Mol. Cell. 2001;7:1245–54.

PLoS Pathog. 2011;7:19–21.

tion. J. Virol. 2002;76:4138–44.

2007;4:63.

82 Advances in Molecular Retrovirology

2004;78:6122.

Virol. 2013;87:12701–20.

1 Infection. J. Virol. 2002;76:8518–31.

within genes. J. Virol. 2006;80:7765–8.

cy Virus Transcription. J Virol. 2005;79:6610–9.

modifications. Genome Res. 2007;17:1186–94.


infected cells and patients by a novel promoter downregulated by Tat. J. Virol. 1994;68:979–87.


[156] Selliah N, Zhang M, DeSimone D, Kim H, Brunner M, Ittenbach RF, et al. The??c-cy‐ tokine regulated transcription factor, STAT5, increases HIV-1 production in primary CD4 T cells. Virology. 2006;344:283–91.

infected cells and patients by a novel promoter downregulated by Tat. J. Virol.

[144] Landry S, Halin M, Lefort S, Audet B, Vaquero C, Mesnard J-M, et al. Detection, characterization and regulation of antisense transcripts in HIV-1. Retrovirology.

[145] Ludwig LB, Ambrus JL, Krawczyk K a, Sharma S, Brooks S, Hsiao C-B, et al. Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing an‐

[146] Kobayashi-Ishihara M, Yamagishi M, Hara T, Matsuda Y, Takahashi R, Miyake A, et al. HIV-1-encoded antisense RNA suppresses viral replication for a prolonged peri‐

[147] Saayman S, Ackley A, Turner A-MW, Famiglietti M, Bosque A, Clemson M, et al. An HIV-encoded antisense long noncoding RNA epigenetically regulates viral transcrip‐

[148] Karn J, Stoltzfus CM. Regulation of HIV-1 Gene Expression. Cold Spring Harb. Per‐

[149] Peeters a, Lambert PF, Deacon NJ. A fourth Sp1 site in the human immunodeficiency virus type 1 long terminal repeat is essential for negative-sense transcription. J. Virol.

[150] Bentley K, Deacon N, Sonza S, Zeichner S, Churchill M. Mutational analysis of the HIV-1 LTR as a promoter of negative sense transcription. Arch. Virol. 2004;149:2277–

[151] Gaynor Richard. Cellular transcription factors involved in the regulation of HIV-1

[152] Rosen C a, Sodroski JG, Haseltine W a. Location of cis-acting regulatory sequences in the human T-cell leukemia virus type I long terminal repeat. Proc. Natl. Acad. Sci. U.

[153] Siekevitz M, Josephs SF, Dukovich M, Peffer N, Wong-Staal F, Greene WC. Activa‐ tion of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I.

[154] Pereira LA, Bentley K, Peeters A, Churchill MJ, Deacon NJ. SURVEY AND SUMMA‐ RY A compilation of cellular transcription factor interactions with the HIV-1 LTR

[155] Kinoshita S, Su L, Amano M, Timmerman L a, Kaneshima H, Nolan GP. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression

tisense RNA and protein products. Retrovirology. 2006;3:80.

1994;68:979–87.

od. Retrovirology. 2012;9:38.

tion. Mol. Ther. 2014;22:1164–75.

gene expression. Aids. 1992. p. 347–63.

spect. Med. 2012;1–17.

1996;70:6665–72.

S. A. 1985;82:6502–6.

Science. 1987;238:1575–8.

promoter. 2000;28:663–8.

in T cells. Immunity. 1997;6:235–44.

94.

2007;4:71.

84 Advances in Molecular Retrovirology


[182] Wang FX, Xu Y, Sullivan J, Souder E, Argyris EG, Acheampong E a., et al. IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of in‐ fected individuals on virally suppressive HAART. J. Clin. Invest. 2005;115:128–37.

[169] Li X, Josef J, Marasco W a. Hiv-1 Tat can substantially enhance the capacity of NIK to induce IkappaB degradation. Biochem. Biophys. Res. Commun. 2001;286:587–94. [170] Pazin MJ, Sheridan PL, Cannon K, Cao Z, Keck JG, Kadonaga JT, et al. NF-κB-medi‐ ated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer

[171] Steger DJ, Workman JL. Stable co-occupancy of transcription factors and histones at

[172] Rothgiesser KM, Erener S, Waibel S, Lüscher B, Hottiger MO. SIRT2 regulates NF-κB dependent gene expression through deacetylation of p65 Lys310. J. Cell Sci.

[173] Kwon H-S, Ott M. The ups and downs of SIRT1. Trends Biochem. Sci. 2008;33:517–25. [174] Zhang JL, Sharma PL, Crumpacker CS. Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein. Virology. 2000;268:251–

[175] Kim YK, Bourgeois CF, Pearson R, Tyagi M, West MJ, Wong J, et al. Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency.

[176] Larochelle S, Amat R, Glover-Cutter K, Sansó M, Zhang C, Allen JJ, et al. Cyclin-de‐ pendent kinase control of the initiation-to-elongation switch of RNA polymerase II.

[177] Takahashi Y, Tanaka Y, Yamashita A. OX40 Stimulation by gp34/OX40 ligand enhan‐ ces prodcuttive human immunodeficiency virus type1 infection. J. Virol.

[178] Kundu M, Srinivasan a, Pomerantz RJ, Khalili K. Evidence that a cell cycle regulator, E2F1, down-regulates transcriptional activity of the human immunodeficiency virus

[179] Majello B, De Luca P, Hagen G, Suske G, Lania L. Different members of the Sp1 mul‐ tigene family exert opposite transcriptional regulation of the long terminal repeat of

[180] Millhouse S, Krebs FC, Yao J, McAllister JJ, Conner J, Ross H, et al. Sp1 and related factors fail to interact with the NF-kappaB-proximal G/C box in the LTR of a replica‐ tion competent, brain-derived strain of HIV-1 (YU-2). J. Neurovirol. 1998;4:312–23.

[181] Garcia-Rodriguez C, Rao A. Nuclear Factor of Activated T Cells (NFAT)-dependent Transactivation Regulated by the Coactivators p300/CREB-binding Protein (CBP). J.

in vitro. Genes Dev. 1996;10:37–49.

2010;123:4251–8.

86 Advances in Molecular Retrovirology

EMBO J. 2006;25:3596–604.

2001;75:674806757.

Nat. Struct. Mol. Biol. 2012;19:1108–15.

type 1 promoter. J. Virol. 1995;69:6940–6.

HIV-1. Nucleic Acids Res. 1994;22:4914–21.

Exp. Med. 1998;187:2031–6.

63.

the HIV-1 enhancer. EMBO J. 1997;16:2463–72.


[206] Agostini I, Navarro JM, Rey F, Bouhamdan M, Spire B, Vigne R, et al. The human immunodeficiency virus type 1 Vpr transactivator: cooperation with promoterbound activator domains and binding to TFIIB. J. Mol. Biol. 1996;261:599–606.

[195] Long J, Wang Y, Wang W, Chang BHJ, Danesh FR. Identification of microRNA-93 as a novel regulator of vascular endothelial growth factor in hyperglycemic conditions.

[196] Novis CL, Archin NM, Buzon MJ, Verdin E, Round JL, Lichterfeld M, et al. Reactiva‐

[197] Pearson R, Kim YK, Hokello J, Lassen K, Friedman J, Tyagi M, et al. Epigenetic si‐ lencing of human immunodeficiency virus (HIV) transcription by formation of re‐ strictive chromatin structures at the viral long terminal repeat drives the progressive

[198] Kim YK, Mbonye U, Hokello J, Karn J. T-cell receptor signaling enhances transcrip‐ tional elongation from latent HIV proviruses by activating P-TEFb through an ERK-

[199] Herrmann CH, Rice a P. Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor. J. Virol.

[200] Wei P, Garber ME, Fang SM, Fischer WH, Jones K a. A novel CDK9-associated Ctype cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-spe‐

[201] Bieniasz PD, Grdina T a, Bogerd HP, Cullen BR. Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat. EM‐

[202] Fujinaga K, Cujec TP, Peng J, Garriga J, Price DH, Graña X, et al. The ability of posi‐ tive transcription elongation factor B to transactivate human immunodeficiency virus transcription depends on a functional kinase domain, cyclin T1, and Tat. J. Virol.

[203] Garber ME, Wei P, KewalRamani VN, Mayall TP, Herrmann CH, Rice AP, et al. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cys‐ teine residue that is not conserved in the murine CycT1 protein. Genes Dev.

[204] Felzien LK, Woffendin C, Hottiger MO, Subbramanian R a, Cohen E a, Nabel GJ. HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300

[205] Wang L, Mukherjee S, Jia F, Narayan O, Zhao LJ. Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and

trans-activation of viral long terminal repeat. J. Biol. Chem. 1995;270:25564–9.

co-activator. Proc. Natl. Acad. Sci. U. S. A. 1998;95:5281–6.

T cells through TLR-1/2 stimulation.

J. Biol. Chem. 2010;285:23457–65.

Retrovirology. 2013;10:119.

88 Advances in Molecular Retrovirology

1995;69:1612–20.

BO J. 1998;17:7056–65.

1998;72:7154–9.

1998;12:3512–27.

tion of latent HIV-1 in central memory CD4+

entry of HIV into latency. J. Virol. 2008;82:12291–303.

dependent pathway. J. Mol. Biol. 2011;410:896–916.

cific binding to TAR RNA. Cell. 1998;92:451–62.


[230] Brиs V, Gomes N, Pickle L, Jones K a. A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat. Genes Dev. 2005;19:1211–26.

[218] Jadlowsky JK, Wong JY, Graham AC, Dobrowolski C, Devor RL, Adams MD, et al. Negative Elongation Factor Is Required for the Maintenance of Proviral Latency but Does Not Induce Promoter-Proximal Pausing of RNA Polymerase II on the HIV

[219] Wagschal A, Rousset E, Basavarajaiah P, Contreras X, Harwig A, Laurent-Chabalier S, et al. Microprocessor, Setx, Xrn2, and Rrp6 co-operate to induce premature termi‐

[220] Kao SY, Calman a F, Luciw P a, Peterlin BM. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987;330:489–93. [221] Laspia MF, Rice a P, Mathews MB. HIV-1 Tat protein increases transcriptional initia‐

[222] Dingwall C, Ernberg I, Gait MJ, Green SM, Heaphy S, Karn J, et al. HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA

[223] Tahirov TH, Babayeva ND, Varzavand K, Cooper JJ, Sedore SC, Price DH. Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature. 2010;465:747–51. [224] Fujinaga K, Irwin D, Huang Y, Taube R, Kurosu T, Peterlin BM. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol. Cell. Biol.

[225] Komarnitsky P, Cho EJ, Buratowski S. Different phosphorylated forms of RNA poly‐ merase II and associated mRNA processing factors during transcription. Genes Dev.

[226] Kim YK, Bourgeois CF, Isel C, Churcher MJ, Karn J. Phosphorylation of the RNA pol‐ ymerase II carboxyl-terminal domain by CDK9 is directly responsible for human im‐ munodeficiency virus type 1 Tat-activated transcriptional elongation. Mol. Cell. Biol.

[227] Czudnochowski N, Bösken C a., Geyer M. Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition. Nat. Commun. 2012;3:842.

[228] Ahn SH, Kim M, Buratowski S. Phosphorylation of serine 2 within the RNA poly‐ merase II C-terminal domain couples transcription and 3Ј end. Process. Mol. Cell.

[229] Lenasi T, Peterlin BM, Barboric M. Cap-binding protein complex links pre-mRNA capping to transcription elongation and alternative splicing through positive tran‐

scription elongation factor b (P-TEFb). J. Biol. Chem. 2011;286:22758–68.

Long Terminal Repeat. Mol. Cell. Biol. 2014;34:1911–28.

nation of transcription by RNAPII. Cell. 2012;150:1147–57.

tion and stabilizes elongation. Cell. 1989;59:283–92.

structure. EMBO J. 1990;9:4145–53.

2004;24:787–95.

90 Advances in Molecular Retrovirology

2000;14:2452–60.

2002;22:4622–37.

2004;13:67–76.


tive transcription elongation factor b and associated proteins in th. Virology. 2000;274:356–66.

[255] Lu H, Li Z, Xue Y, Schulze-Gahmen U, Johnson JR, Krogan NJ, et al. AFF1 is a ubiq‐ uitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and for‐ mation of SECs for HIV transactivation. Proc. Natl. Acad. Sci.. 2014;111 :E15–24.

[242] Markert A, Grimm M, Martinez J, Wiesner J, Meyerhans A, Meyuhas O, et al. The Larelated protein LARP7 is a component of the 7SK ribonucleoprotein and affects tran‐

scription of cellular and viral polymerase II genes. EMBO Rep. 2008;9:569–75.

[243] Schröder S, Cho S, Zeng L, Zhang Q, Kaehlcke K, Mak L, et al. Two-pronged binding with bromodomain-containing protein 4 liberates positive transcription elongation factor b from inactive ribonucleoprotein complexes. J. Biol. Chem. 2012;287:1090–9.

[244] Bisgrove D a, Mahmoudi T, Henklein P, Verdin E. Conserved P-TEFb-interacting do‐ main of BRD4 inhibits HIV transcription. Proc. Natl. Acad. Sci. U. S. A.

[245] Jang MK, Mochizuki K, Zhou M, Jeong H-S, Brady JN, Ozato K. The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA poly‐

[246] Yang Z, Yik JHN, Chen R, He N, Jang MK, Ozato K, et al. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol.

[247] Zhu J, Gaiha GD, John SP, Pertel T, Chin CR, Gao G, et al. Reactivation of latent

[248] Wu S-Y, Chiang C-M. The double bromodomain-containing chromatin adaptor Brd4

[249] Boehm D, Calvanese V, Dar RD, Xing S, Schroeder S, Martins L, et al. BET bromodo‐ main-targeting compounds reactivate HIV from latency via a Tat-independent mech‐

[250] Denis G V, McComb ME, Faller D V, Sinha A, Romesser PB, Costello CE. Identifica‐ tion of transcription complexes that contain the double bromodomain protein Brd2

[251] Cherrier T, Le Douce V, Eilebrecht S, Riclet R, Marban C, Dequiedt F, et al. CTIP2 is a negative regulator of P-TEFb. Proc. Natl. Acad. Sci. U. S. A. 2013;110:12655–60.

[252] Mbonye UR, Wang B, Gokulrangan G, Chance MR, Karn J. Phosphorylation of HEX‐ IM1 at Tyr271 and Tyr274 Promotes Release of P-TEFb from the 7SK snRNP Com‐ plex and Enhances Proviral HIV Gene Expression. Proteomics. 2015;15:2078–86.

[253] Ji X, Zhou Y, Pandit S, Huang J, Li H, Lin CY, et al. SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase. Cell.

[254] Suñé C, Goldstrohm a C, Peng J, Price DH, Garcia-Blanco M a. An in vitro transcrip‐ tion system that recapitulates equine infectious anemia virus tat-mediated inhibition of human immunodeficiency virus type 1 Tat activity demonstrates a role for posi‐

and chromatin remodeling machines. J. Proteome Res. 2006;5:502–11.

merase II-dependent transcription. Mol. Cell. 2005;19:523–34.

HIV-1 by inhibition of BRD4. Cell Rep. 2012;2:807–16.

and transcriptional regulation. J. Biol. Chem. 2007;282:13141–5.

2007;104:13690–5.

92 Advances in Molecular Retrovirology

Cell. 2005;19:535–45.

2013;153:855–68.

anism. Cell Cycle. 2013;12:452–62.


[280] Van Lint C, Emiliani S, Ott M, Verdin E. Transcriptional activation and chromatin re‐ modeling of the HIV-1 promoter in response to histone acetylation. EMBO J. 1996;15:1112–20.

[268] Col E, Caron C, Seigneurin-Berny D, Gracia J, Favier A, Khochbin S. The Histone Acetyltransferase, hGCN5, Interacts with and Acetylates the HIV Transactivator, Tat.

[269] Dorr A, Kiermer V, Pedal A, Rackwitz HR, Henklein P, Schubert U, et al. Transcrip‐ tional synergy between Tat and PCAF is dependent on the binding of acetylated Tat

[270] Kaehlcke K, Dorr A, Hetzer-Egger C, Kiermer V, Henklein P, Schnoelzer M, et al. Acetylation of Tat defines a CyclinT1-independent step in HIV transactivation. Mol.

[271] Mahmoudi T, Parra M, Vries RGJ, Kauder SE, Verrijzer CP, Ott M, et al. The SWI/SNF chromatin-remodeling complex is a cofactor for Tat transactivation of the

[272] Tréand C, du Chéné I, Brès V, Kiernan R, Benarous R, Benkirane M, et al. Require‐ ment for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the

[273] Agbottah E, Deng L, Dannenberg LO, Pumfery A, Kashanchi F. Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription. Retrovirology.

[274] Pagans S, Pedal A, North BJ, Kaehlcke K, Marshall BL, Dorr A, et al. SIRT1 regulates

[275] Pagans S, Kauder SE, Kaehlcke K, Sakane N, Schroeder S, Dormeyer W, et al. The Cellular lysine methyltransferase Set7/9-KMT7 binds HIV-1 TAR RNA, monomethy‐ lates the viral transactivator Tat, and enhances HIV transcription. Cell Host Microbe.

[276] Sakane N, Kwon HS, Pagans S, Kaehlcke K, Mizusawa Y, Kamada M, et al. Activa‐ tion of hiv transcription by the viral tat protein requires a demethylation step medi‐ ated by lysine-specific demethylase 1 (LSD1/KDM1). PLoS Pathog. 2011;7:1–12. [277] Brès V, Kiernan RE, Linares LK, Chable-Bessia C, Plechakova O, Tréand C, et al. A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 pro‐

[278] Verdin E. DNase I-hypersensitive sites are associated with both long terminal repeats and with the intragenic enhancer of integrated human immunodeficiency virus type

[279] Verdin E, Paras P, Van Lint C. Chromatin disruption in the promoter of human im‐ munodeficiency virus type 1 during transcriptional activation. EMBO J.

HIV transcription via Tat deacetylation. PLoS Biol. 2005;3:0210–20.

J. Biol. Chem. 2001;276:28179–84.

Cell. 2003;12:167–76.

94 Advances in Molecular Retrovirology

2006;3:48.

2010;7:234–44.

to the PCAF bromodomain. EMBO J. 2002;21:2715–23.

HIV promoter. J. Biol. Chem. 2006;281:19960–8.

HIV-1 promoter. EMBO J. 2006;25:1690–9.

moter. Nat. Cell Biol. 2003;5:754–61.

1. J. Virol. 1991;65:6790–9.

1993;12:3249–59.


[304] Witwer KW, Watson AK, Blankson JN, Clements JE. Relationships of PBMC micro‐ RNA expression, plasma viral load, and CD4+ T-cell count in HIV-1-infected elite suppressors and viremic patients. Retrovirology. 2012;9:5.

[292] Bouchat S, Gatot J-S, Kabeya K, Cardona C, Colin L, Herbein G, et al. Histone meth‐ yltransferase inhibitors induce HIV-1 recovery in resting CD4(+) T cells from HIV-1-

[293] Du Chéné I, Basyuk E, Lin Y-L, Triboulet R, Knezevich A, Chable-Bessia C, et al. Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcrip‐

[294] Friedman J, Cho W-K, Chu CK, Keedy KS, Archin NM, Margolis DM, et al. Epigenet‐ ic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste

[295] Imai K, Togami H, Okamoto T. Involvement of histone H3 lysine 9 (H3K9) methyl‐ transferase G9a in the maintenance of HIV-1 latency and its reactivation by BIX01294.

[296] Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of

[297] Blazkova J, Trejbalova K, Gondois-Rey F, Halfon P, Philibert P, Guiguen A, et al. CpG methylation controls reactivation of HIV from latency. PLoS Pathog. 2009;5. [298] Palacios JA, Pérez-Piñar T, Toro C, Sanz-Minguela B, Moreno V, Valencia E, et al. Long-term nonprogressor and elite controller patients who control viremia have a higher percentage of methylation in their HIV-1 proviral promoters than aviremic patients receiving highly active antiretroviral therapy. J. Virol. 2012;86:13081–4. [299] Triboulet R, Mari B, Lin Y, Chable-bessia C, Bennasser Y, Lebrigand K, et al. Suppres‐ sion of MicroRNA-Silencing Pathway by HIV-1 During Virus Replication. Science

[300] Nathans R, Chu C-Y, Serquina AK, Lu C-C, Cao H, Rana TM. Cellular microRNA and P bodies modulate host-HIV-1 interactions. Mol. Cell. Elsevier Ltd; 2009;34:696–

[301] Imam H, Shahr Bano A, Patel P, Holla P, Jameel S. The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by

[302] Wang X, Ye L, Hou W, Zhou Y, Wang Y-J, Metzger DS, et al. Cellular microRNA ex‐ pression correlates with susceptibility of monocytes/macrophages to HIV-1 infection.

[303] Houzet L, Yeung ML, de Lame V, Desai D, Smith SM, Jeang K-T. MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1) seropositive individuals.

early and late viral proteins. Sci. Rep. 2015;5:8639.

HIV-1 latency by cytosine methylation. PLoS Pathog. 2009;5:e1000495.

tional silencing and post-integration latency. EMBO J. 2007;26:424–35.

infected HAART-treated patients. AIDS. 2012;26:1473–82.

2. J. Virol. 2011;85:9078–89.

96 Advances in Molecular Retrovirology

(80-.). 2007;1579–82.

Blood. 2009;113:671–4.

Retrovirology. 2008;5:118.

709.

J. Biol. Chem. 2010;285:16538–45.


[331] Whisnant AW, Bogerd HP, Flores O, Ho P, Powers JG, Sharova N, et al. In-depth analysis of the interaction of HIV-1 with cellular microRNA biogenesis and effector mechanisms. MBio. 2013;4:e000193.

[317] Sun G, Li H, Wu X, Covarrubias M, Scherer L, Meinking K, et al. Interplay between HIV-1 infection and host microRNAs. Nucleic Acids Res. 2012;40:2181–96.

[318] Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: Tar‐

[319] Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, et al. A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing. Cell.

[320] Ahluwalia JK, Khan SZ, Soni K, Rawat P, Gupta A, Hariharan M, et al. Human cellu‐ lar microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1

[321] Ruelas DS, Chan JK, Oh E, Heidersbach AJ, Hebbeler AM, Chavez L, et al. Micro‐ RNA-155 Reinforces HIV Latency. J. Biol. Chem. 2015;290:jbc.M115.641837.

[322] Zhang HS, Chen XY, Wu TC, Sang WW, Ruan Z. MiR-34a is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation through the SIRT1/NF??B pathway.

[323] Zhang H-S, Wu T-C, Sang W-W, Ruan Z. MiR-217 is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation by down-regulation of SIRT1. Biochim. Bi‐

[324] Chen XY, Zhang HS, Wu TC, Sang WW, Ruan Z. Down-regulation of NAMPT ex‐ pression by miR-182 is involved in Tat-induced HIV-1 long terminal repeat (LTR)

[325] Ma L, Shen CJ, Cohen É a., Xiong SD, Wang JH. MiRNA-1236 inhibits HIV-1 infec‐ tion of monocytes by repressing translation of cellular factor VprBP. PLoS One.

[326] Bennasser Y, Le S-Y, Yeung ML, Jeang K-T. HIV-1 encoded candidate micro-RNAs

[327] Yeung ML, Houzet L, Yedavalli VSRK, Jeang K-T. A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1

[328] Schopman NCT, Willemsen M, Liu YP, Bradley T, Van Kampen A, Baas F, et al. Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs. Nucleic Acids

[329] Ouellet DL, Plante I, Landry P, Barat C, Janelle M-E, Flamand L, et al. Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR

[330] Omoto S, Fujii YR. Regulation of human immunodeficiency virus 1 transcription by

transactivation. Int. J. Biochem. Cell Biol. Elsevier B.V.; 2013;45:292–8.

and their cellular targets. Retrovirology. 2004;1:43.

replication. J. Biol. Chem. 2009;284:19463–73.

element. Nucleic Acids Res. 2008;36:2353–65.

nef microRNA. J. Gen. Virol. 2005;86:751–5.

FEBS Lett. Federation of European Biochemical Societies; 2012;586:4203–7.

gets and expression. Nucleic Acids Res. 2008;36:149–53.

2009;129:1401–14.

98 Advances in Molecular Retrovirology

replication. Retrovirology. 2008;5:117.

ophys. Acta. 2012;1823:1017–23.

2014;9:1–7.

Res. 2012;40:414–27.


on suppressive antiretroviral therapy at concentrations achieved by clinical dosing. PLoS Pathog. Public Library of Science; 2014;10:e1004071.


[356] Moody MA, Santra S, Vandergrift NA, Sutherland LL, Gurley TC, Drinker MS, et al. Toll-like receptor 7/8 (TLR7/8) and TLR9 agonists cooperate to enhance HIV-1 enve‐ lope antibody responses in rhesus macaques. J. Virol. 2014;88:3329–39.

on suppressive antiretroviral therapy at concentrations achieved by clinical dosing.

[345] Archin NM, Liberty AL, Kashuba AD, Choudhary SK, Kuruc JD, Crooks AM, et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral

[346] Archin NM, Bateson R, Tripathy MK, Crooks AM, Yang K-H, Dahl NP, et al. HIV-1 expression within resting CD4+ T cells after multiple doses of vorinostat. J. Infect.

[347] Bartholomeeusen K, Xiang Y, Fujinaga K, Peterlin BM. Bromodomain and extra-ter‐ minal (BET) bromodomain inhibition activate transcription via transient release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonu‐

[348] Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. Nature Pub‐ lishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.;

[349] Xing S, Siliciano RF. Targeting HIV latency: pharmacologic strategies toward eradi‐

[350] Bernhard W, Barreto K, Saunders A, Dahabieh MS, Johnson P, Sadowski I. The Suv39H1 methyltransferase inhibitor chaetocin causes induction of integrated HIV-1

[351] Fernandez G, Zeichner SL. Cell line-dependent variability in HIV activation employ‐

[352] Blazkova J, Murray D, Justement JS, Funk EK, Nelson AK, Moir S, et al. Paucity of HIV DNA methylation in latently infected, resting CD4+ T cells from infected indi‐

[353] Thibault S, Imbeault M, Tardif MR, Tremblay MJ. TLR5 stimulation is sufficient to trigger reactivation of latent HIV-1 provirus in T lymphoid cells and activate virus

[354] Chang JJ, Altfeld M. Immune activation and the role of TLRs and TLR agonists in the pathogenesis of HIV-1 infection in the humanized mouse model. J. Infect. Dis.

[355] De Jong MAWP, de Witte L, Oudhoff MJ, Gringhuis SI, Gallay P, Geijtenbeek TBH. TNF-alpha and TLR agonists increase susceptibility to HIV-1 transmission by human Langerhans cells ex vivo. J. Clin. Invest. American Society for Clinical Investigation;

gene expression in central memory CD4+ T cells. Virology. 2009;389:20–5.

PLoS Pathog. Public Library of Science; 2014;10:e1004071.

therapy. Nature. 2012;487:482–5.

cleoprotein. J. Biol. Chem. 2012;287:36609–16.

ing DNMT inhibitors. Virol. J. 2010;7:266.

2013;208 Suppl :S145–9.

2008;118:3440–52.

cation. Drug Discov. Today. Elsevier Ltd; 2013;18:541–51.

without producing a T cell response. FEBS Lett. 2011;585:3549–54.

viduals receiving antiretroviral therapy. J. Virol. 2012;86:5390–2.

Dis. 2014;210:728–35.

100 Advances in Molecular Retrovirology

2014;13:673–91.


[380] Hazuda D, Barnard R, Wolkenberg S, Powell D, Karn J, Das B, et al. HIV latency drug discovery: Optimizing drugs to induce latent HIV expression. Proc. 6th Int. Work. HIV Persistence Dur. Ther. Miami, FL, USA, 3–6 December 2013. 2013;

[368] Choudhary SK, Archin NM, Margolis DM. Hexamethylbisacetamide and disruption of human immunodeficiency virus type 1 latency in CD4(+) T cells. J. Infect. Dis.

[369] Klichko V, Archin N, Kaur R, Lehrman G, Margolis D. Hexamethylbisacetamide re‐ models the human immunodeficiency virus type 1 (HIV-1) promoter and induces Tat-independent HIV-1 expression but blunts cell activation. J. Virol. 2006;80:4570–9.

[370] Pérez M, de Vinuesa AG, Sanchez-Duffhues G, Marquez N, Bellido ML, Muñoz-Fer‐ nandez MA, et al. Bryostatin-1 synergizes with histone deacetylase inhibitors to reac‐

[371] Biancotto A, Grivel J-C, Gondois-Rey F, Bettendroffer L, Vigne R, Brown S, et al. Du‐ al role of prostratin in inhibition of infection and reactivation of human immunodefi‐ ciency virus from latency in primary blood lymphocytes and lymphoid tissue. J.

[372] Wang P, Qu X, Wang X, Liu L, Zhu X, Zeng H, et al. As2O3 synergistically reactivate

[373] Mochly-Rosen D, Khaner H, Lopez J. Identification of intracellular receptor proteins

[374] Trushin SA, Bren GD, Asin S, Pennington KN, Paya C V, Badley AD. Human immu‐ nodeficiency virus reactivation by phorbol esters or T-cell receptor ligation requires

[375] Colin L, Vandenhoudt N, de Walque S, Van Driessche B, Bergamaschi A, Martinelli V, et al. The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for viral replication. PLoS One. Public Library of Science;

[376] Hirsch I, Caux C, Hasan U, Bendriss-Vermare N, Olive D. Impaired Toll-like receptor 7 and 9 signaling: from chronic viral infections to cancer. Trends Immunol.

[377] Chang JJ, Altfeld M. Immune activation and the role of TLRs and TLR agonists in the pathogenesis of HIV-1 infection in the humanized mouse model. J. Infect. Dis.

[378] Gallastegui E, Marshall B, Vidal D, Sanchez-Duffhues G, Collado JA, Alvarez-Fer‐ nández C, et al. Combination of biological screening in a cellular model of viral laten‐ cy and virtual screening identifies novel compounds that reactivate HIV-1. J. Virol.

[379] Das B, Dobrowolski C, Mao H, Powell D, Miller M, Hazuda D, et al. Farnesyl trans‐ ferase: A new target for eradication of latent HIV-1 provirus in jurkat T-cells. Proc. 6th Int. Work. HIV Persistence Dur. Ther. Miami, FL, USA, 3–6 December 2013. 2013.

latent HIV-1 by induction of NF-κB. Antiviral Res. 2013. p. 688–97.

for activated protein kinase C. Proc. Natl. Acad. Sci. 1991;88:3997–4000.

tivate HIV-1 from latency. Curr. HIV Res. 2010;8:418–29.

both PKCalpha and PKCtheta. J. Virol. 2005;79:9821–30.

2008;197:1162–70.

102 Advances in Molecular Retrovirology

Virol. 2004;78:10507–15.

2011;6:e19084.

2010;31:391–7.

2013;208 Suppl :S145–9.

2012;86:3795–808.


[406] Shingai M, Nishimura Y, Klein F, Mouquet H, Donau OK, Plishka R, et al. Antibodymediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature. 2013;503:277–80.

[392] Ye F, Karn J. Bacterial Short Chain Fatty Acids Push All The Buttons Needed To Re‐

[393] Shirakawa K, Chavez L, Hakre S, Calvanese V, Verdin E. Reactivation of latent HIV by histone deacetylase inhibitors. Trends Microbiol. Elsevier Ltd; 2013;21:277–85.

[394] Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. Viral persis‐ tence alters CD8 T-cell immunodominance and tissue distribution and results in dis‐

[395] Khaitan A, Unutmaz D. Revisiting immune exhaustion during HIV infection. Curr.

[396] Palmer BE, Neff CP, Lecureux J, Ehler A, Dsouza M, Remling-Mulder L, et al. In vivo blockade of the PD-1 receptor suppresses HIV-1 viral loads and improves CD4+ T

[397] (NIAID) NI of A and ID. Safety and Immune Response of BMS-936559 in HIV-Infect‐

[398] Migueles SA, Osborne CM, Royce C, Compton AA, Joshi RP, Weeks KA, et al. Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associat‐

[399] Shan L, Deng K, Shroff NS, Durand CM, Rabi SA, Yang H-C, et al. Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir

[400] Hansen SG, Ford JC, Lewis MS, Ventura AB, Hughes CM, Coyne-Johnson L, et al. Profound early control of highly pathogenic SIV by an effector memory T-cell vac‐

[401] Hansen SG, Piatak M, Ventura AB, Hughes CM, Gilbride RM, Ford JC, et al. Immune

[402] Hansen SG, Sacha JB, Hughes CM, Ford JC, Burwitz BJ, Scholz I, et al. Cytomegalovi‐ rus vectors violate CD8+ T cell epitope recognition paradigms. Science.

[403] Barouch DH, Whitney JB, Moldt B, Klein F, Oliveira TY, Liu J, et al. Therapeutic effi‐ cacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected

[404] Barouch DH, Stephenson KE, Borducchi EN, Smith K, Stanley K, McNally AG, et al. Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV chal‐

[405] Klein F, Halper-Stromberg A, Horwitz JA, Gruell H, Scheid JF, Bournazos S, et al. HIV therapy by a combination of broadly neutralizing antibodies in humanized

clearance of highly pathogenic SIV infection. Nature. 2013;502:100–4.

activate Latent Viruses. Stem cell epigenetics. 2015;2.

HIV/AIDS Rep. 2011;8:4–11.

104 Advances in Molecular Retrovirology

cine. Nature. 2011;473:523–7.

rhesus monkeys. Nature.; 2013;503:224–8.

mice. Nature. 2012;492:118–22.

lenges in rhesus monkeys. Cell. 2013;155:531–9.

2013;340:1237874.

tinct stages of functional impairment. J. Virol. 2003;77:4911–27.

cell levels in humanized mice. J. Immunol. 2013;190:211–9.

ed People Taking Combination Antiretroviral Therapy. 2015.

ed with immune control. Immunity. 2008;29:1009–21.

after virus reactivation. Immunity. 2012;36:491–501.


**Retroviral Infection Prevention Strategies**

## **Which Vaccination Strategies and Immune Responses are More Effective Against HIV Infections?**

Azam Bolhassani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61913

#### **Abstract**

Vaccination is one of the most successful approaches for controlling various viral diseas‐ es. Novel approaches will be needed to develop highly effective vaccines to prevent infec‐ tious diseases such as HIV. There are many aspects of HIV-1 biology that make the development of an HIV vaccine difficult, including viral diversity, effective type of im‐ mune response, and suitable experimental model for preclinical trials. In spite of these challenges, recent published results showed that a vaccine regimen could reduce HIV in‐ fection rates by 31% in Thailand. This vaccine named as RV144 is composed of a recombi‐ nant canarypox vector expressing three HIV-1 proteins as a prime and two different recombinant HIV-1 gp120 envelope glycoproteins with alum adjuvant as a boost. In addi‐ tion, a subunit vaccine constructed from the viral envelope protein could be efficiently developed using new techniques available through genetic engineering. The current HIV-1 vaccine development focuses on antibody-based approaches. It was shown that immunization with the viral envelope glycoprotein, gp120, should generate neutralizing antibodies that would prevent infection, thereby yielding protective immunity. However, HIV could develop many pathways to escape from antibodies that bind to the different parts of the viral envelope molecules. Thus, the generation of neutralizing antibodies is very difficult after viral infection or immunization protocols. Indeed, the viral envelope molecules (Env) possess glycosylated residues that cover surface epitopes for binding and neutralizing antibodies, even if the antibodies are produced. Furthermore, the tri‐ meric structures of envelope molecules show rapid conformational changes due to the in‐ teraction with viral cell surface receptors, CCR5/CXCR4 and CD4; thus the transition state is very poor to be recognized by the immune system. Currently, studies focus on generating stable trimeric envelope molecules (gp120/gp41) as immunogens that can in‐ duce neutralizing antibodies that can compete for binding to the cell surface receptors. Altogether, it is clear that the design of a vaccine to elicit HIV-neutralizing antibodies is not straightforward, and it causes major challenges in structural biology and immunolo‐ gy, several other studies strongly suggest cytotoxic T-lymphocyte (CTL)-based immune responses against HIV infections. Indeed, CD8+ T cells play a major role in controlling vi‐ ral replication during primary HIV infections and in maintaining a stable viral load dur‐ ing the chronic phase. In this line, live-attenuated vaccines could elicit more potent and durable pathogen-specific immune responses than inactivated or subunit vaccines. Gen‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

erally, DNA vaccines are poorly immunogenic alone, and viral vector vaccines are inef‐ fective due to vector-specific immune responses if used repeatedly; hence, the two approaches have often been tested in combination as prime-boost vaccination strategies. Indeed, the prime-boost vaccination has been considered as an efficient strategy against HIV infections. In this chapter, we will represent challenges to determine the best vaccine strategies against HIV infections.

**Keywords:** HIV infection, Immune responses, Vaccination, DNA vaccine, Prime-boost vaccine, Adjuvant, Challenges for HIV vaccine

#### **1. Introduction**

#### **1.1. HIV infection and vaccination**

According to recent reports, 35 million people were living with HIV-1 at the end of 2013, the considerable majority being in Sub-Saharan Africa, with dynamic epidemics in Asia [1]. HIV infection results in gradual loss of CD4+ T lymphocytes, containing immune competence, and progression to AIDS. Effective treatment with combined antiretroviral drugs decreases viral load below detectable levels but cannot eliminate the virus from the body. Furthermore, the success of combined antiretroviral drugs is hindered by accumulating drug toxicities and chronic immune activation leading to increased risk of several non-AIDS disorders, even when viral replication is inhibited. Therefore, there is a major need for therapeutic strategies as an alternative to the combined antiretroviral drugs [2]. HIV vaccine strategies are expected to be a critical component for controlling the HIV epidemic [3, 4]. Immunotherapy, or therapeutic vaccination, aims to enhance existing immune responses against HIV or stimulate immune responses. These immune responses should provide an efficient cure by controlling viral replication and preventing disease progression in the absence of combined antiretroviral drugs [2]. The cost-effective and different HIV-1 vaccine approaches have recently attracted a special interest. Both antibodies and cell-mediated immune responses are considered to be important to prevent HIV-1 infection in the mucosal compartment, *i.e.*, the entry point for sexual transmission [1]. A great majority of HIV-1 infections occur at mucosa during sexual contact. Therefore, it is important to provide mucosal barrier protection against this entry by mucosal vaccination. A number of mucosal routes of vaccination such as enteric oral or intranasal vaccines have significant barriers that limit vaccine efficacy or cause safety risks. In contrast, the sublingual region of the mouth could provide a simple route for mucosal vaccination with immunogens, but this site does not always induce strong immune responses, especially when protein antigens are used [5].

Currently, antibody-inducing vaccines are a major focus in the preventive HIV vaccine field [6]. In addition, T-cell-based therapeutic vaccines have focused on three strategies: a) to increase the levels of vaccine-induced responses, b) to enhance the responses targeting only conserved regions of the virus, and c) to use replication-competent viral vectors [1]. Generally, antiviral T-cell and B-cell responses play a crucial role in suppressing HIV replication during chronic infection [7, 8]. Novel approaches of HIV treatment include both conventional therapeutic vaccines (*i.e.*, active immunization strategies using HIV-derived immunogens) and the use of checkpoint blockers such as anti-PD-1 antibodies. These complex therapeutic strategies appear as promising approaches against HIV infection [7].

erally, DNA vaccines are poorly immunogenic alone, and viral vector vaccines are inef‐ fective due to vector-specific immune responses if used repeatedly; hence, the two approaches have often been tested in combination as prime-boost vaccination strategies. Indeed, the prime-boost vaccination has been considered as an efficient strategy against HIV infections. In this chapter, we will represent challenges to determine the best vaccine

**Keywords:** HIV infection, Immune responses, Vaccination, DNA vaccine, Prime-boost

According to recent reports, 35 million people were living with HIV-1 at the end of 2013, the considerable majority being in Sub-Saharan Africa, with dynamic epidemics in Asia [1]. HIV

progression to AIDS. Effective treatment with combined antiretroviral drugs decreases viral load below detectable levels but cannot eliminate the virus from the body. Furthermore, the success of combined antiretroviral drugs is hindered by accumulating drug toxicities and chronic immune activation leading to increased risk of several non-AIDS disorders, even when viral replication is inhibited. Therefore, there is a major need for therapeutic strategies as an alternative to the combined antiretroviral drugs [2]. HIV vaccine strategies are expected to be a critical component for controlling the HIV epidemic [3, 4]. Immunotherapy, or therapeutic vaccination, aims to enhance existing immune responses against HIV or stimulate immune responses. These immune responses should provide an efficient cure by controlling viral replication and preventing disease progression in the absence of combined antiretroviral drugs [2]. The cost-effective and different HIV-1 vaccine approaches have recently attracted a special interest. Both antibodies and cell-mediated immune responses are considered to be important to prevent HIV-1 infection in the mucosal compartment, *i.e.*, the entry point for sexual transmission [1]. A great majority of HIV-1 infections occur at mucosa during sexual contact. Therefore, it is important to provide mucosal barrier protection against this entry by mucosal vaccination. A number of mucosal routes of vaccination such as enteric oral or intranasal vaccines have significant barriers that limit vaccine efficacy or cause safety risks. In contrast, the sublingual region of the mouth could provide a simple route for mucosal vaccination with immunogens, but this site does not always induce strong immune responses, especially when

Currently, antibody-inducing vaccines are a major focus in the preventive HIV vaccine field [6]. In addition, T-cell-based therapeutic vaccines have focused on three strategies: a) to increase the levels of vaccine-induced responses, b) to enhance the responses targeting only conserved regions of the virus, and c) to use replication-competent viral vectors [1]. Generally, antiviral T-cell and B-cell responses play a crucial role in suppressing HIV replication during chronic infection [7, 8]. Novel approaches of HIV treatment include both conventional

T lymphocytes, containing immune competence, and

strategies against HIV infections.

**1.1. HIV infection and vaccination**

protein antigens are used [5].

infection results in gradual loss of CD4+

**1. Introduction**

110 Advances in Molecular Retrovirology

vaccine, Adjuvant, Challenges for HIV vaccine

The biggest barrier for many vaccines is the pathogen's variability. Thus, studies should be further focused on the functionally most conserved regions of proteins common to many variants, including escape mutants inducing both antibody and T-cell immune responses. For vector-based vaccines, the "universal" subunit immunogens are efficiently delivered using heterologous prime-boost regimens, which can be further improved using adjuvants and delivery approaches [9–11]. Several studies have described the development of vaccine strategies, including improved envelope proteins formulated with potent adjuvants, DNA and vectors expressing mosaics or conserved sequences, capable of inducing strong relevant immune responses, such as neutralizing antibodies (NAbs) and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immuno‐ logical memory. The type of immune response elicited by different immunogens can also correlate with the risk of HIV infections. For example, IgG antibodies against the V2 loop of gp120 are associated with a decreased risk of HIV infection, while Env-specific IgA antibody is directly related to increased risk [12]. Generally, a combination of two independent ap‐ proaches, containing the induction of broadly neutralizing antibodies (bNAbs) to prevent or reduce acquisition of infection and stimulation of effective CTL responses, is the currently used technique to slow disease progression in advance infections [13].

Briefly, more than 20 years after the discovery of HIV, researchers are trying to design a protective AIDS vaccine. The problem is the lack of basic knowledge about the immunological requirements for the protection against HIV. Virus diversity and escape from immune responses are the most important challenges to the development of an effective HIV vaccine. In this chapter, we will represent the challenges to vaccine design against HIV biologically and immunologically. Moreover, different vaccine strategies will be described to determine the best strategies already focused on HIV infections. In this line, the relationship between HIV biology and immunity will be demonstrated for the first time.

#### **2. Immunogen-induced Neutralizing Antibodies (NAbs)**

The most common tests for HIV infections rely on detecting antibodies against virus. Thus, these tests can also detect antibodies induced by a candidate HIV vaccine. The detection of vaccine-induced antibodies to HIV by serological tests is referred to as vaccine-induced seroreactivity (VISR) [6]. Neutralizing antibodies are useful in identifying the neutralizing epitopes of vaccine and for understanding the mechanism of potent and broad cross-neutral‐ ization, thereby providing a modality of preventive and therapeutic value [14, 15]. It has been shown that some NAbs confer protection toward neonatal HIV-1 infection [16].

Various types of HIV-1 Env immunogens were developed that express epitopes for broadly neutralizing antibodies and their precursors. There are three new structures proposed for the HIV-1 Env trimer, which will be more immunogenic than previously used immunogens: a) minimal immunogens that are fragments of HIV-1 Env-neutralizing epitopes, b) intermediate Env immunogens (*e.g.*, monomeric Env gp120), and c) various forms of Env trimers [17]. To date, these structures have not been capable of inducing the immune system to generate bNAbs after vaccination. Thus, a successful vaccination for HIV-1 and induction of bNAbs will need repetitive immunizations for a long time [17].

The current studies of HIV-positive patients with strongly neutralizing sera indicated that the immune system is able to produce antibodies neutralizing up to 90% of HIV strains [18]. The neutralizing antibodies bind to conserved gp120 sites, and the identification of these sites can help to design effective vaccines. Glycosylated residues (or carbohydrates) have a key role because of binding broadly neutralizing antibodies to carbohydrates and combining carbohydrate and peptide elements on gp120. However, carbohydrates partial‐ ly cover some peptides on envelope surface recognized by bNAbs. Thus, the use of engineered glycoproteins as vaccines for the stimulation of bNAbs is a subject of interest in HIV vaccine design [18].Antibody responses to the HIV-1 envelope glycoproteins can be classified into two: a) non-neutralizing responses directed to peptide epitopes expressed on isolated envelope glycoproteins but not on the native envelope trimers responsible for mediating the entry of virus into target cells; b) broadly neutralizing antibody responses targeting epitopes expressed on the native envelope trimers. Currently, many potent broadly neutralizing antibodies have been isolated to stimulate prophylactic and therapeutic activities in animal models. These antibodies help us to improve vaccine design and therapeutic strategies for HIV-1 [19]. The recent characterization of new epitopes for stimulating broadly neutralizing antibodies has encouraged studies in the synthesis of novel antigenic constructs for the development of HIV-envelope-directed vaccines [20]. Thus, an important step in vaccine design is the determination of antibodies and epitopes associat‐ ed with broad HIV neutralization. Indeed, immunogens and/or immunization protocols should be designed to increase antibody affinity maturation [1].

Regarding the studies, HIV-1 envelope gp120 is the target for neutralizing antibodies against the virus. HIV-1 envelope gp120 exhibits a great degree of variability that causes a major challenge for the development of vaccines against HIV/AIDS. Different approaches have been used to improve immunogenicity of broadly neutralizing epitopes on HIV-1 gp120 with limited success [21]. For example, immunogenicity of gp120 and its V3 epitopes was enhanced when gp120 was co-injected with monoclonal antibodies (mAb) to the CD4-binding sites (CD4bs). Indeed, the gp120/CD4bs complex was a potent immunogen for eliciting crossreactive functional NAbs against V3 epitopes [21]. In contrast, the membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) includes epitopes for recognizing bNAb as an important region in vaccine development. However, designing an immunogen for the induction of bNAbs to MPER is difficult because of the relative inaccessibility of the MPER in the native conformation of Env [22]. Therefore, a group of oligomeric gp41 immunogens was designed to further expose MPER in a suitable conforma‐ tion. The immunogens comprised different gp41 N-heptad lengths and insertion of extra epitopes and flexible C-termini. These immunogens were used in two different immunization strategies, including gp41/gp140 proteins and gp41/gp160 DNA associated with various adjuvants and modalities. It was observed that the gp41 immunogens elicit higher levels of MPER than the gp140 immunogens. In prime-boost strategies, the best MPER responses were shown in the groups receiving gp41 DNA followed by gp41 protein. Several agents may influence MPER immunogenicity such as the immunization route, dose, or adjuvant. Gener‐ ally, these data encourage the researchers for designing MPER immunogens with optimized immunization protocols [22]. Furthermore, the aggregation of HIV-1 virions was detected by antibodies (IgG) to the viral envelope glycoprotein (Env). Neutralizing antibodies directed to a V3-base- and glycan-dependent epitope on gp120 and to the apex of the Env trimer, as well as non-neutralizing antibodies to the epitope cluster I on the gp41-ectodomain, could aggre‐ gate virions, but the neutralizing antibody 2G12, which is specific for a glycan-dependent monovalent epitope on gp120, could not aggregate. These data can potentially open the ways for the development of HIV-1 vaccine [23].

#### **3. Preventive HIV vaccines**

minimal immunogens that are fragments of HIV-1 Env-neutralizing epitopes, b) intermediate Env immunogens (*e.g.*, monomeric Env gp120), and c) various forms of Env trimers [17]. To date, these structures have not been capable of inducing the immune system to generate bNAbs after vaccination. Thus, a successful vaccination for HIV-1 and induction of bNAbs will need

The current studies of HIV-positive patients with strongly neutralizing sera indicated that the immune system is able to produce antibodies neutralizing up to 90% of HIV strains [18]. The neutralizing antibodies bind to conserved gp120 sites, and the identification of these sites can help to design effective vaccines. Glycosylated residues (or carbohydrates) have a key role because of binding broadly neutralizing antibodies to carbohydrates and combining carbohydrate and peptide elements on gp120. However, carbohydrates partial‐ ly cover some peptides on envelope surface recognized by bNAbs. Thus, the use of engineered glycoproteins as vaccines for the stimulation of bNAbs is a subject of interest in HIV vaccine design [18].Antibody responses to the HIV-1 envelope glycoproteins can be classified into two: a) non-neutralizing responses directed to peptide epitopes expressed on isolated envelope glycoproteins but not on the native envelope trimers responsible for mediating the entry of virus into target cells; b) broadly neutralizing antibody responses targeting epitopes expressed on the native envelope trimers. Currently, many potent broadly neutralizing antibodies have been isolated to stimulate prophylactic and therapeutic activities in animal models. These antibodies help us to improve vaccine design and therapeutic strategies for HIV-1 [19]. The recent characterization of new epitopes for stimulating broadly neutralizing antibodies has encouraged studies in the synthesis of novel antigenic constructs for the development of HIV-envelope-directed vaccines [20]. Thus, an important step in vaccine design is the determination of antibodies and epitopes associat‐ ed with broad HIV neutralization. Indeed, immunogens and/or immunization protocols

Regarding the studies, HIV-1 envelope gp120 is the target for neutralizing antibodies against the virus. HIV-1 envelope gp120 exhibits a great degree of variability that causes a major challenge for the development of vaccines against HIV/AIDS. Different approaches have been used to improve immunogenicity of broadly neutralizing epitopes on HIV-1 gp120 with limited success [21]. For example, immunogenicity of gp120 and its V3 epitopes was enhanced when gp120 was co-injected with monoclonal antibodies (mAb) to the CD4-binding sites (CD4bs). Indeed, the gp120/CD4bs complex was a potent immunogen for eliciting crossreactive functional NAbs against V3 epitopes [21]. In contrast, the membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) includes epitopes for recognizing bNAb as an important region in vaccine development. However, designing an immunogen for the induction of bNAbs to MPER is difficult because of the relative inaccessibility of the MPER in the native conformation of Env [22]. Therefore, a group of oligomeric gp41 immunogens was designed to further expose MPER in a suitable conforma‐ tion. The immunogens comprised different gp41 N-heptad lengths and insertion of extra epitopes and flexible C-termini. These immunogens were used in two different immunization strategies, including gp41/gp140 proteins and gp41/gp160 DNA associated with various

repetitive immunizations for a long time [17].

112 Advances in Molecular Retrovirology

should be designed to increase antibody affinity maturation [1].

The studies indicated that a successful vaccine candidate needs to elicit broad antibodies targeting the Env protein. Immunogens targeting gp120 have been developed, which block infection in monkeys. Attempts to induce antibody persistence were complicated by increasing the number of HIV-target cells [24]. RV144 consisting of canary pox vector vaccine ALVAC-HIV (vCP1521) prime and AIDSVAX®gp120 B/E boost was the first vaccine against HIV-1 infection achieved in clinical trial [1, 25]. The analysis of vaccine-induced immune responses in vaccinated-infected and vaccinated-uninfected volunteers indicated that IgG specific for the V1V2 region of gp120 was related to the decreased risk of HIV-1 infection, and plasma Env IgA was directly associated with infection risk. Thus, RV144 studies indicated that Env is essential and possibly sufficient to stimulate protective antibody responses against mucosally acquired HIV-1. Efficacy trials were planned in heterosexual populations in southern Africa and Thailand [1]. Generally, the studies of nonhuman primates suggest that Env is a necessary component for a successful protection against viral infections. Two approaches are being followed to induce Env-specific-antibody-mediated protection: a) vaccines that elicit potent and broadly reactive neutralizing antibodies against viruses which are common in human transmission, b) vaccines that induce antibody neutralizing only in less commonly transmitted HIV strains, but that block HIV-1 infection by non-neutralizing (nNAbs) mechanisms [26, 27]. Monomeric gp120 HIV-1 envelope proteins alone failed to protect high-risk individuals against infection. In fact, the level and breadth of elicited NAb were not sufficient for protection [1]. Furthermore, the results indicated that IgG to linear epitopes in the V2 and V3 regions of gp120 is part of a complex interaction of immune responses that contribute to the protection in RV144 [28]. In RV144, Env IgG3 was correlated with decreased risk of HIV infection, a response that declined rapidly compared to overall IgG responses. Indeed, the rates of Envspecific IgG3 and V1/V2 IgG3 responses were high, and conversely IgG4 responses were considerably low in recipients of the RV144 vaccine. These findings indicated that V2 IgG plays a role in protection against HIV-1 infection. Generally, an increase in magnitude, affinity, breadth, and importantly in frequency and durability of V2- and V3-specific antibodies of IgG3 and IgG1 subclasses may confer a higher and more durable rate of protection against HIV-1 infection. The induction of cross-reactive V1V2-specific IgG raises the hypothesis of cross-clade protection. Additional booster vaccinations may increase the antibody levels. Residual antibody responses against gp120 were detected 6–8 years post vaccination in RV144 vaccinees. Additional boosts increased plasma IgG gp120 and gp70 V1/V2 antibodies at titers higher than those in RV144, while weak gp120 IgA responses were induced. These HIV-specific IgG antibodies were also detected in rectal secretions while IgAs were undetectable [29–34].

Regarding the studies, HIV antibodies capable of preventing mucosal cell-free or cell-to-cell HIV transmission are critical for the development of effective prophylactic and therapeutic vaccines. The interactions between antigen-presenting cells (APCs) and HIV result in cell-tocell transmission of HIV. In the experimental macaque model, data indicated that the broadly neutralizing antibodies are capable of neutralizing an extensive range of HIV strains, prevent‐ ing cell-to-cell transfer, and protecting from infection[35]. In addition, IgG Fcg receptor (FcgR) mediated inhibition of antibodies at the mucosal site may play a role in protection against HIV mucosal transmission. On the contrary, mucosal IgA antibodies may be effective in protection against HIV sexual transmission. Thus, the determination of inhibitory effects of antibodies is critical for evaluating protection in HIV vaccines [35]. Furthermore, the majority of the antibodies against different viral proteins described a marker (shared idiotope) that is recognized by the monoclonal antibody 1F7. This shared idiotype on antibodies induced by HIV-1 was involved in the immune memory mechanism linking the early and late antibodies, the so-called back-boost effect. This finding was supported by auto-antibodies that bind to the 1F7 idiotope in sera of HIV-1-infected individuals. The expression of a shared idiotope in antibodies could provide a strategy to stimulate B cells selected to produce antibodies against HIV-1 and HCV, suggesting their implications in vaccine design [36].

The use of potent adjuvants may also enhance antigen-specific antibody responses. Several adjuvants have been tested in nonhuman primates and humans indicating a significant benefit of HIV envelope proteins formulated with MF59 and AS01 adjuvants. A study indicated that alum protected macaques from simian immunodeficiency virus (SIV)mac251 infection, while MF59 did not protect despite its ability to elicit higher systemic T-cell and antibody responses. Adjuvant-associated differences in the homing of plasmablasts and induction of key cellular signaling pathways may explain these effects. The formulation of HIV-1 gp120 with MPLA and alum induced significantly higher levels of neutralizing antibodies and T-cell lympho‐ proliferation compared to alum, MF59, or MPLA alone. Importantly, antibodies to gp70 V1V2 (subtypes B, C, and CRF01 AE) were induced more rapidly, to a higher magnitude and with a greater durability than alum-adjuvanted gp120 [37–42]. Also, the formulation of antigens with solid nanoparticles may prolong the duration of antibody responses by increasing antigen retention locally in the tissues driving B-cell responses, enhancing dendritic cell (DC) antigen presentation, and the development of CD4+ Th cells that provide cytokines and signals that are required to initiate somatic hypermutation and affinity maturation for effective B-cell memory [1]. Furthermore, the ability of two mucosal adjuvants, including α-galactosylcera‐ mide (α-GalCer) as a potent stimulator of natural killer (NK) T cells and CpG-oligodeoxynu‐ cleotide (CpG-ODN) as a TLR9 agonist, was evaluated to enhance immune responses against clade C gp140 HIV-1 envelope protein antigen. The results showed that CD4+ and CD8+ T-cell responses in systemic and mucosal tissues were significantly higher in mice immunized with gp140 in the presence of either α-GalCer or CpG-ODN and further enhanced when both adjuvants were used. Also, the use of two adjuvants and especially their combination effec‐ tively increased gp140-specific serum IgG and vaginal IgA antibody levels. Memory T-cell responses detected at 60 days after immunization revealed that α-GalCer is more potent than CpG-ODN, and the combination of α-GalCer and CpG-ODN adjuvants was more effective than either alone [5]. Another approach is called B-cell lineage vaccine design. In this line, the recombinant antibodies belonging to bNAb members were used to determine HIV-1 envelope constructs as immunogens in a prime-boost strategy. These envelope constructs utilize the naïve B-cell repertoire residing in bone marrow and secondary lymphoid tissues *in vivo* and subsequently stimulate B-cell evolution until bNAb-producing cells are elicited [43, 44].

and IgG1 subclasses may confer a higher and more durable rate of protection against HIV-1 infection. The induction of cross-reactive V1V2-specific IgG raises the hypothesis of cross-clade protection. Additional booster vaccinations may increase the antibody levels. Residual antibody responses against gp120 were detected 6–8 years post vaccination in RV144 vaccinees. Additional boosts increased plasma IgG gp120 and gp70 V1/V2 antibodies at titers higher than those in RV144, while weak gp120 IgA responses were induced. These HIV-specific IgG antibodies were also detected in rectal secretions while IgAs were undetectable [29–34].

Regarding the studies, HIV antibodies capable of preventing mucosal cell-free or cell-to-cell HIV transmission are critical for the development of effective prophylactic and therapeutic vaccines. The interactions between antigen-presenting cells (APCs) and HIV result in cell-tocell transmission of HIV. In the experimental macaque model, data indicated that the broadly neutralizing antibodies are capable of neutralizing an extensive range of HIV strains, prevent‐ ing cell-to-cell transfer, and protecting from infection[35]. In addition, IgG Fcg receptor (FcgR) mediated inhibition of antibodies at the mucosal site may play a role in protection against HIV mucosal transmission. On the contrary, mucosal IgA antibodies may be effective in protection against HIV sexual transmission. Thus, the determination of inhibitory effects of antibodies is critical for evaluating protection in HIV vaccines [35]. Furthermore, the majority of the antibodies against different viral proteins described a marker (shared idiotope) that is recognized by the monoclonal antibody 1F7. This shared idiotype on antibodies induced by HIV-1 was involved in the immune memory mechanism linking the early and late antibodies, the so-called back-boost effect. This finding was supported by auto-antibodies that bind to the 1F7 idiotope in sera of HIV-1-infected individuals. The expression of a shared idiotope in antibodies could provide a strategy to stimulate B cells selected to produce antibodies against

The use of potent adjuvants may also enhance antigen-specific antibody responses. Several adjuvants have been tested in nonhuman primates and humans indicating a significant benefit of HIV envelope proteins formulated with MF59 and AS01 adjuvants. A study indicated that alum protected macaques from simian immunodeficiency virus (SIV)mac251 infection, while MF59 did not protect despite its ability to elicit higher systemic T-cell and antibody responses. Adjuvant-associated differences in the homing of plasmablasts and induction of key cellular signaling pathways may explain these effects. The formulation of HIV-1 gp120 with MPLA and alum induced significantly higher levels of neutralizing antibodies and T-cell lympho‐ proliferation compared to alum, MF59, or MPLA alone. Importantly, antibodies to gp70 V1V2 (subtypes B, C, and CRF01 AE) were induced more rapidly, to a higher magnitude and with a greater durability than alum-adjuvanted gp120 [37–42]. Also, the formulation of antigens with solid nanoparticles may prolong the duration of antibody responses by increasing antigen retention locally in the tissues driving B-cell responses, enhancing dendritic cell (DC) antigen

are required to initiate somatic hypermutation and affinity maturation for effective B-cell memory [1]. Furthermore, the ability of two mucosal adjuvants, including α-galactosylcera‐ mide (α-GalCer) as a potent stimulator of natural killer (NK) T cells and CpG-oligodeoxynu‐ cleotide (CpG-ODN) as a TLR9 agonist, was evaluated to enhance immune responses against

Th cells that provide cytokines and signals that

HIV-1 and HCV, suggesting their implications in vaccine design [36].

presentation, and the development of CD4+

114 Advances in Molecular Retrovirology

The nNAb B-cell progenitors become activated and internalize Env compared with bNAb Bcell progenitors. The reports showed that rational immunogen modifications can reduce the activation of naïve B cells that lead to such nNAbs, while promoting the activation of naïve B cells that result in germline-reverted bNAbs [43, 44]. A number of potent broadly neutralizing antibodies have been identified from HIV-infected individuals although the generation of bNAbs using traditional vaccine approaches has been obscure. The researchers tested a single dose of 3BNC117 or 10-1074 (mAb specific for the CD4-binding site and the V3 region, respectively) and also a combination of both antibodies. The data showed a total decline in viral loads post infusion in simian-human immunodeficiency virus (SHIV)-infected macaques. Another approach to the generation of bNAbs is to circumvent "normal" immune responses and direct non-lymphoid cells to produce bNAbs *in vivo* using gene therapy. Vectored immunoprophylaxis (VIP) is a gene therapy method in which transgenes encoding bNAbs are delivered directly into muscle tissue where bNAbs are produced. Two recent animal studies demonstrated that VIP could generate modest titers of NAb that can effectively prevent *in vivo* HIV infection in a humanized bone marrow–thymus–liver (BLT) HIV infection model and a simian immunodeficiency macaque infection model [7]. Recent findings showed that adenoassociated virus (AAV)-delivered broadly neutralizing antibodies can inhibit HIV replication. Indeed, a single injection of AAV could generate long-term antibody responses as a therapeutic approach in the lack of antiretroviral drugs. Induction of vector-mediated antibodies could inhibit cell-to-cell transmission and replication of HIV. This result represented an alternative to immunogen-based vaccine design and a novel therapeutic intervention by enabling particular manipulation of humoral immunity [45, 46].

A challenge for HIV-1 immunogen design is the induction of neutralizing antibodies against neutralization-resistant (Tier-2) viruses that control human transmissions. A soluble recombi‐ nant HIV-1 envelope glycoprotein trimer possessing a native conformation (BG505 SOSIP.664) could induce NAbs potently against the sequence-matched Tier-2 virus in rabbits but weaker and similar responses in macaques. The trimer also stably stimulated cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies, whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, nativelike trimers may represent a promising starting point for developing HIV-1 vaccines directed at inducing bNAbs [47].

The goal of an HIV vaccine is to generate robust and durable protective antibody. Thus, it is important to induce CD4+ T-follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and the quality of vaccine-elicited antibody titers [48]. In this line, a DNA/modified *vaccinia* virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide was administered in rhesus macaques. The studies indicated that booster immunization with modified *vaccinia* virus Ankara induces a distinct and transient accumulation of proliferating CXCR5+ and CXCR5<sup>−</sup> CD4 T cells in blood at day 7 post-immunization, and the frequency of the former but not the latter correlated with TFH and B-cell responses in germinal centers of the lymph node. Furthermore, gp140 boost elicited a skewing toward CXCR3 expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential of vaccine-elicited antibody response. However, CXCR3+ cells preferentially expressed the HIV co-receptor CCR5, and vaccine-induced CXCR3+ CXCR5+ cells showed a moderate positive association with peak viremia following SIV251 infection. These data demonstrated that vaccine regimens eliciting CXCR3-biased TFH cell responses favor antibody persistence and avidity but may prompt higher acute viremia in advance infections [48, 49].

Generally, broadly neutralizing antibodies specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for the protection against multiple HIV-1 clades. Recently, an HIV vaccine incorporating the molecular adjuvants B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) was designed with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. The results indicated that mice immunization with a DNA vaccine encoding BAFF or APRIL multitrimers, together with IL-12 and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against Tier-1 and Tier-2 viruses. The APRIL-containing vaccine was especially effective at generating Tier-2 neutralizing antibodies following a protein boost. Notably, BAFF and APRIL did not cause B-cell expansion or an increase in total IgG. Thus, BAFF and APRIL multitrimers were proposed as promising molecular adjuvants for inducing bNAbs against HIV-1 infection [50].

#### **4. Stimulation of cell-mediated immune responses**

A successful HIV vaccine must either completely prevent infection or eliminate the first round of infected CD4 T cells before the latent pool of HIV-infected cells is established. Thus, an effective HIV vaccine requires high levels of protective immunity at the time of virus contact with the host, and it cannot rely on memory immune responses to occur. CD8 T cells can effectively kill HIV-infected T cells, but in most cases of acute HIV infection, the virus rapidly escapes. For example, anti-HIV CD8 CTL activity is capable of eliminat‐ ing virus-infected T cells in the setting of vaccination with an attenuated rhesus cytomega‐ lovirus (rhCMV) containing simian immunodeficiency virus genes, but in the setting of acute HIV infection, the transmitted/founder virus usually escapes from CD8 T-cell control [51]. CTL responses targeting specific HIV proteins (*e.g.*, Gag) have been associated with relative control of viral replication *in vivo*. In a SIVmac251 intravenous challenge model, breadth of Gag CTL epitope recognition correlated with control of viremia peak [1]. These data have demonstrated that CD8+ T cells are associated with the control and eradication of early retrovirus infections [17].

B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, nativelike trimers may represent a promising starting point for developing HIV-1 vaccines directed

The goal of an HIV vaccine is to generate robust and durable protective antibody. Thus, it is important to induce CD4+ T-follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and the quality of vaccine-elicited antibody titers [48]. In this line, a DNA/modified *vaccinia* virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide was administered in rhesus macaques. The studies indicated that booster immunization with modified *vaccinia* virus

CD4 T cells in blood at day 7 post-immunization, and the frequency of the former but not the latter correlated with TFH and B-cell responses in germinal centers of the lymph node. Furthermore, gp140 boost elicited a skewing toward CXCR3 expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential

association with peak viremia following SIV251 infection. These data demonstrated that vaccine regimens eliciting CXCR3-biased TFH cell responses favor antibody persistence and

Generally, broadly neutralizing antibodies specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for the protection against multiple HIV-1 clades. Recently, an HIV vaccine incorporating the molecular adjuvants B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) was designed with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. The results indicated that mice immunization with a DNA vaccine encoding BAFF or APRIL multitrimers, together with IL-12 and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against Tier-1 and Tier-2 viruses. The APRIL-containing vaccine was especially effective at generating Tier-2 neutralizing antibodies following a protein boost. Notably, BAFF and APRIL did not cause B-cell expansion or an increase in total IgG. Thus, BAFF and APRIL multitrimers were proposed as promising

A successful HIV vaccine must either completely prevent infection or eliminate the first round of infected CD4 T cells before the latent pool of HIV-infected cells is established. Thus, an effective HIV vaccine requires high levels of protective immunity at the time of virus contact with the host, and it cannot rely on memory immune responses to occur. CD8 T cells can effectively kill HIV-infected T cells, but in most cases of acute HIV infection, the virus rapidly escapes. For example, anti-HIV CD8 CTL activity is capable of eliminat‐ ing virus-infected T cells in the setting of vaccination with an attenuated rhesus cytomega‐

CXCR5+

and CXCR5<sup>−</sup>

cells preferentially expressed the HIV

cells showed a moderate positive

Ankara induces a distinct and transient accumulation of proliferating CXCR5+

avidity but may prompt higher acute viremia in advance infections [48, 49].

molecular adjuvants for inducing bNAbs against HIV-1 infection [50].

**4. Stimulation of cell-mediated immune responses**

of vaccine-elicited antibody response. However, CXCR3+

co-receptor CCR5, and vaccine-induced CXCR3+

at inducing bNAbs [47].

116 Advances in Molecular Retrovirology

The vaccine trials were focused on whether cell-mediated immune-response-inducing vaccines can prevent infection or reduce post-infection plasma viral load [1]. Vaccinees with HLA alleles associated with HIV-1 control had a significantly lower mean viral load over time. Interestingly, the most highly conserved epitopes were detected at a lower frequen‐ cy, suggesting that stronger responses to conserved sequences may be as important as breadth for protection [52, 53]. Interestingly, heterologous vector prime-boost regimens enhanced immunity by increasing the magnitude, onset, and multi-functionality of the insert-specific cell-mediated immune responses compared to homologous regimens [54]. New progress has been made in overcoming HIV-1 diversity through induction of crossreactive T-cell responses to HIV-1 by vaccines designed *in silico* (called conserved and mosaic vaccines) [17]. Polyvalent mosaic immunogens derived by the recombination of natural HIV-1 strains were designed to induce cellular immune responses that recognize genetically diverse circulating virus isolates. Increasing the breadth and depth of epitope recognition may contribute to the protection against infection by genetically diverse viruses and also to the control of variant viruses that emerge as they mutate away from recogni‐ tion by CTLs. For example, mosaic HIV-1 Gag, Pol, and Env antigens expressed by Ad26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T-lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys [55, 56]. An alternative to multiva‐ lent wild-type or mosaic vaccines is the use of conserved element immunogens as a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. Priming with conserved elements boosted with the complete immunogen induced broad cellular and humoral immunity focused on the conserved regions of the virus. In contrast, full-length HIV-1 immunogens elicited greater magnitude and compara‐ ble breadth of T-lymphocyte responses to conserved HIV-1 regions compared with conserved-region only HIV-1 immunogens in rhesus monkeys [57, 58].

An important point is the use of replicating vector. A replication-competent rhesus cytome‐ galovirus vaccine expressing SIV proteins induced and maintained high frequency of SIVspecific CD4+ and CD8+ T-cell effector memory responses at extra-lymphoid sites without measurable antibody responses to SIV. Half of vaccinated monkeys showed a severe control of three routes of SIVmac239 transmission including intrarectal, intravaginal, and intravenous. The conservation of particular cytotoxic epitopes does make them good candidates for a global HIV-1 vaccine [59, 60].

#### **5. Therapeutic HIV vaccines**

Recent studies have focused on the improvement of effective prophylactic and therapeu‐ tic approaches to combat persistent viral infections. Therapeutic vaccines for HIV infec‐ tion should aim to elicit antiviral CD8 T cells (CTLs), CD4 T cells, and neutralizing antibody since these immune responses control viral replication [7, 61]. It is critical to generate broad cellular responses as HIV mutates very rapidly to escape immune system. In addition, recent studies determined that T-follicular helper cells constitute a significant source of virus production and contribute to the total viral reservoir. Since these cells reside in B-cell follicles/germinal centers, it may be critical to generate CD8 T cells that can home to Bcell follicles and exert immune response on these cells. The HIV-specific CD4 T-cell response is also important for maintaining the functional CD8 T-cell and B-cell responses. Howev‐ er, these HIV-specific CD4 T cells could also serve as potential targets for virus replica‐ tion [7]. Interestingly, CD4 T cells with cytolytic function have been shown to be associated with enhanced viral control, although it is demonstrated whether these responses can be primed by vaccination. The function of dendritic cells may be also critical for generating a protective cellular and humoral immune response, as chronic HIV infections are associat‐ ed with impaired DC function. Thus, therapeutic vaccines may also need to use strategies such as adjuvants to enhance the function of innate immunity [7].

Several therapeutic vaccine strategies have already been used such as live-attenuated mi‐ crobes, viral vectors, and dendritic cell-based vaccines that led to suppress and/or clear infections. Among them, improved DNA vaccines have emerged as a promising candidate for the treatment of infectious diseases especially HIV infections [61–63]. Some strategies have been considered to improve immune responses stimulated by DNA vaccines such as *in vivo* efficient DNA delivery systems, co-delivery with molecular adjuvants as well as the develop‐ ment of potent heterologous prime-boost regimens [61, 62, 64]. DNA vaccines have been utilized as candidate HIV vaccines because of their ability to generate cellular and humoral immune responses, the lack of anti-vector response allowing for repeat administration, and their ability to prime the response to viral-vectored vaccines. Because the HIV epidemic has unreasonably affected the developing world, the favorable thermostability profile and relative ease and low cost of manufacture of DNA vaccines offer additional advantages. *In vivo* electroporation (EP) has been utilized to improve immune responses to DNA vaccines as candidate HIV-1 vaccines alone or prime-boost regimens with both proteins and viral-vectored vaccines in several animal models, and recently, in human clinical trials [65]. In addition, intradermal electroporation of HIV DNA was well tolerated. Strong cell- and antibodymediated immune responses were elicited by the HIV-DNA prime and HIV-Modified Vaccinia Ankara (MVA) boosting regimen, with or without intradermal electroporation use [66]. DNA vaccines have an intrinsic bias toward generating cellular immunity against intracellular pathogens. By manipulating the DNA formulation and delivery, effective antibody responses can also be induced. For instance, studies showed that the immunized monkeys with DNA vaccine developed HIV-specific T-cell immune responses that persisted for months [62].

The use of live-attenuated invasive bacteria as a carrier for DNA-based vaccines has previously been reported [67]. Immunization with recombinant invasive bacteria including *Shigella*, *Salmonella*, and *Listeria* carrying plasmid DNA (pDNA) vaccines has been shown to induce protective immune responses in mice. The use of human enteric bacteria is especially useful due to their ability to infect human colonic mucosa, and their tropism for the activation of dendritic cells and macrophage of internal mucosa. Thus, they are very efficient for the delivery of DNA vaccines to APCs in the mucosa resulting in stimulation of potent systemic and local immune responses. Such responses may be critical for the development of an effective prophylactic HIV vaccine, because a large number of HIV transfer through human mucosal routes [67]. For instance, a live-attenuated strain of *Salmonella typhimurium* was used to deliver plasmid orally and showed an adjuvant role through the release of various cytokines [68]. In addition, intranasal immunization of mice with live recombinant *Shigella* cells induced an HIV Gag-specific cellular immune response similar to that observed by intramuscular injection of naked DNA. Importantly, a strong boosting effect was obtained in mice primed with DNA, suggesting the efficacy of bacterial vectors in prime-boost vaccination regimens [67]. The studies indicated that a novel vaccine delivery system using bacterial ghosts (BGs) can be considered as an efficient and nontoxic delivery system for DNA vaccines *in vitro* and *in vivo*. In this line, a new strategy of HIV vaccine delivery was designed using *Salmonella typhi* Ty21a bacterial ghosts. The data showed that Ty21a BGs loaded with an HIV gp140 DNA vaccine (Ty21a BG-DNA) are easily taken up by murine macrophage cells (RAW264.7), and gp140 is efficiently expressed in these cells. Peripheral and intestinal mucosal anti-gp120 antibody responses in mice vaccinated with BGs–DNA vaccine were significantly higher than those in mice immunized with naked DNA vaccine. The enhancement of antibody responses was associated with BG-induced production of IL-10 through TLR4 pathway [69].

**5. Therapeutic HIV vaccines**

118 Advances in Molecular Retrovirology

Recent studies have focused on the improvement of effective prophylactic and therapeu‐ tic approaches to combat persistent viral infections. Therapeutic vaccines for HIV infec‐ tion should aim to elicit antiviral CD8 T cells (CTLs), CD4 T cells, and neutralizing antibody since these immune responses control viral replication [7, 61]. It is critical to generate broad cellular responses as HIV mutates very rapidly to escape immune system. In addition, recent studies determined that T-follicular helper cells constitute a significant source of virus production and contribute to the total viral reservoir. Since these cells reside in B-cell follicles/germinal centers, it may be critical to generate CD8 T cells that can home to Bcell follicles and exert immune response on these cells. The HIV-specific CD4 T-cell response is also important for maintaining the functional CD8 T-cell and B-cell responses. Howev‐ er, these HIV-specific CD4 T cells could also serve as potential targets for virus replica‐ tion [7]. Interestingly, CD4 T cells with cytolytic function have been shown to be associated with enhanced viral control, although it is demonstrated whether these responses can be primed by vaccination. The function of dendritic cells may be also critical for generating a protective cellular and humoral immune response, as chronic HIV infections are associat‐ ed with impaired DC function. Thus, therapeutic vaccines may also need to use strategies

Several therapeutic vaccine strategies have already been used such as live-attenuated mi‐ crobes, viral vectors, and dendritic cell-based vaccines that led to suppress and/or clear infections. Among them, improved DNA vaccines have emerged as a promising candidate for the treatment of infectious diseases especially HIV infections [61–63]. Some strategies have been considered to improve immune responses stimulated by DNA vaccines such as *in vivo* efficient DNA delivery systems, co-delivery with molecular adjuvants as well as the develop‐ ment of potent heterologous prime-boost regimens [61, 62, 64]. DNA vaccines have been utilized as candidate HIV vaccines because of their ability to generate cellular and humoral immune responses, the lack of anti-vector response allowing for repeat administration, and their ability to prime the response to viral-vectored vaccines. Because the HIV epidemic has unreasonably affected the developing world, the favorable thermostability profile and relative ease and low cost of manufacture of DNA vaccines offer additional advantages. *In vivo* electroporation (EP) has been utilized to improve immune responses to DNA vaccines as candidate HIV-1 vaccines alone or prime-boost regimens with both proteins and viral-vectored vaccines in several animal models, and recently, in human clinical trials [65]. In addition, intradermal electroporation of HIV DNA was well tolerated. Strong cell- and antibodymediated immune responses were elicited by the HIV-DNA prime and HIV-Modified Vaccinia Ankara (MVA) boosting regimen, with or without intradermal electroporation use [66]. DNA vaccines have an intrinsic bias toward generating cellular immunity against intracellular pathogens. By manipulating the DNA formulation and delivery, effective antibody responses can also be induced. For instance, studies showed that the immunized monkeys with DNA vaccine developed HIV-specific T-cell immune responses that persisted for months [62].

such as adjuvants to enhance the function of innate immunity [7].

Attenuated virus vaccines have traditionally been potent and relatively easy to produce and deliver. Vaccination with a live virus results in high intracellular synthesis of viral proteins. This high-level expression stimulates strong cellular and humoral immune responses and results in the production of long-lasting memory B and T cells. However, attenuated HIV vaccines replicate strongly in animal models to retain residual virulence. Recent studies indicated that priming with a DNA vaccine induces a Th1 response that can be boosted by the subsequent administration of a viral vector encoding the same gene. This prime-boost strategy elicited strong protective immunity in several primate models [70]. Currently, immunogenicity of a highly attenuated *vaccinia* virus with low neuro-virulence, LC16m8 strain, was studied as an HIV vaccine vector. The data showed that the recombinant *vaccinia* virus-based vaccine (vLC-Env) combined with DNA vaccine expressing the HIV *env* gene (pCAG-Env) produces a protective immune response against HIV infection in BALB/c mice. Vaccination of vLC-Env alone induced much higher HIV-specific humoral and cellular immune responses than that of pCAG-Env. Priming with pCAG-Env further enhanced vLC-Env-induced immune responses, especially cell-mediated immune response. In addition, administration of vLC-Env-infected dendritic cells to mice generated a high cellular immune response. These results demonstrated that priming with pCAG-Env and boosting with vLC-Env represents a potential candidate for vaccination against HIV infection [70]. A few studies have used DC presenting either the autologous virus or virus-derived peptides as therapeutic vaccines (DC-based vaccines) in macaques and humans. They also showed that a similar approach could successfully control HIV replication in humans. Similarly, a recent study showed that an efficient HIV-1-specific immune response could be generated using an autologous monocyte-derived DC (MDDC) transfer. Thus, HIV-specific immune responses could be elicited by DC-based therapeutic vaccinations. In general, therapeutic vaccinations should be explored as a combination therapy with other immune modulators to achieve a functional cure (*i.e.*, long-term control of virus replication in the absence of antiretroviral therapy) [7].

There is growing interest in the role of anti-HIV antibody-dependent cellular cytotoxicity (ADCC) antibodies in the prevention and control of HIV infection. Passive transfer studies in macaques supported a role for the Fc region of antibodies in the prevention of simian–human immunodeficiency virus infection. The Thai RV144 HIV-1 vaccine trial induced anti-HIV ADCC antibodies that may play a role in the partial protection observed. Several studies showed a role for ADCC antibodies in slowing HIV disease progression. However, HIV evolves to escape ADCC antibodies, and chronic HIV infections cause the dysfunction of effector cells such as natural killer cells that mediate the ADCC functions. Furthermore, four recent studies showed that the HIV-1 *Vpu* protein, by promoting release of virions, reduces the capacity of ADCC antibodies to recognize HIV-infected cells [71]. On the contrary, The HIV-1 transactivator of transcription (Tat) is a key HIV virulence factor, which plays critical roles in virus gene expression, replication, transmission, and disease progression. The results indicated that Tat-induced immune responses are necessary to restore immune homeostasis, to block the replenishment and to reduce the size of the viral reservoir. Anti-Tat antibodies are uncommon in natural infection and, when present, correlate with the asymptomatic state and lead to lower or no disease progression. Hence, targeting Tat represents a pathogenesis-driven intervention [72].

#### **6. Adjuvants**

Although the importance of DNA vaccines, especially as a priming immunization, has been well proved in different HIV vaccine studies, the immunogenicity of DNA vaccines is generally moderate. Novel adjuvant is necessary for improving the immunogenicity of DNA vaccine [73]. Multiple groups have demonstrated the potential of co-administering plasmid DNA that express cytokines, chemokines, or co-stimulatory molecules together with plasmids encoding target viral antigens [74]. The potency of DNA vaccines can be developed by the co-delivery of plasmid-encoded molecular adjuvants. The pDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), hematopoietic factor fms-like tyrosine kinase 3 ligand (Flt-3L), and interleukin-12 (IL-12) could markedly enhance cell-mediated immune responses elicited by an HIV-1 *env* pDNA vaccine in BALB/c mice [74]. Plasmid GM-CSF also increased the immune responses elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pGM-CSF as a vaccine adjuvant appeared to increase antigen-specific proliferative responses and the percentage of polyfunctional memory CD8+ T cells. Co-delivery of pFlt-3L with pGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pGM-CSF with pIL-12 significantly enhanced Env-specific prolifera‐ tive responses and vaccine efficacy in the murine *vaccinia* virus challenge model relative to mice immunized with the *env* pDNA vaccine adjuvanted with either pGM-CSF or pIL-12 alone [74]. In another study, co-administration of the HIV-1 DNA vaccine with pIL-12 and pGM-CSF by topical application to the skin enhanced the levels of both the HIV-specific cytotoxic T-lymphocyte response and delayed-type hypersensitivity (DTH). Indeed, the skin is accessi‐ ble for generating immune responses by both intradermal injection and topical use of gene delivery vectors [75]. In addition, co-administration of plasmids encoding the codon-opti‐ mized GM-CSF sequence with the HIV-1 Gag DNA vaccine resulted in a strong antibody and CTL response and a protective immune response against infection with recombinant *vaccinia* virus expressing HIV-1 Gag [76]. On the contrary, researchers strongly support the use of IL-6 or IL-15 as a cytokine adjuvant in HIV DNA vaccination. The data indicated that intranasal administration of DNA vaccine and pIL-15 can enhance Th1-dependent HIV-1-specific cellmediated immunity. However, co-injection of pIL-15 with pIL-2 or pIL-12 did not show any synergistic effect on the immune responses induced by DNA vaccine *in vivo* [77]. Furthermore, the immunogenicity of HIV-1 DNA vaccine expressing the chimeric gene Gag-gp120 (pVAX1- Gag-gp120) was increased by co-inoculating pVAX1-IL6 in BALB/c mice [78]. The studies demonstrated that in-frame fusion of tumor necrosis factor alpha (TNF-α), DNA to DNA, encoding a large fragment of HIV gp120 could enhance Th1 immune responses against gp120 antigen. Also, in-frame fusion of IFNγ-encoding DNA at the 5′ end of the chimeric molecule, to create a tripartite fusion, had no additional effect on immunogenicity [79]. A number of studies have shown that α-galactosylceramide, a natural killer T-cell (NKT) ligand, was applied as an adjuvant for various vaccines, including viral, parasite, and protein-based vaccines. The α-GalCer was able to enhance HIV-specific antibody responses. Furthermore, co-administration of α-GalCer with suboptimal doses of DNA vaccines greatly increased antigen-specific CD4+ and CD8+ T-cell responses. The level of cell-mediated immune responses in mice vaccinated with 5 μg of DNA in the presence of α-GalCer was similar to that of mice vaccinated with 50 μg of DNA in the absence of α-GalCer [80].

macaques and humans. They also showed that a similar approach could successfully control HIV replication in humans. Similarly, a recent study showed that an efficient HIV-1-specific immune response could be generated using an autologous monocyte-derived DC (MDDC) transfer. Thus, HIV-specific immune responses could be elicited by DC-based therapeutic vaccinations. In general, therapeutic vaccinations should be explored as a combination therapy with other immune modulators to achieve a functional cure (*i.e.*, long-term control of virus

There is growing interest in the role of anti-HIV antibody-dependent cellular cytotoxicity (ADCC) antibodies in the prevention and control of HIV infection. Passive transfer studies in macaques supported a role for the Fc region of antibodies in the prevention of simian–human immunodeficiency virus infection. The Thai RV144 HIV-1 vaccine trial induced anti-HIV ADCC antibodies that may play a role in the partial protection observed. Several studies showed a role for ADCC antibodies in slowing HIV disease progression. However, HIV evolves to escape ADCC antibodies, and chronic HIV infections cause the dysfunction of effector cells such as natural killer cells that mediate the ADCC functions. Furthermore, four recent studies showed that the HIV-1 *Vpu* protein, by promoting release of virions, reduces the capacity of ADCC antibodies to recognize HIV-infected cells [71]. On the contrary, The HIV-1 transactivator of transcription (Tat) is a key HIV virulence factor, which plays critical roles in virus gene expression, replication, transmission, and disease progression. The results indicated that Tat-induced immune responses are necessary to restore immune homeostasis, to block the replenishment and to reduce the size of the viral reservoir. Anti-Tat antibodies are uncommon in natural infection and, when present, correlate with the asymptomatic state and lead to lower or no disease progression. Hence, targeting Tat represents a pathogenesis-driven

Although the importance of DNA vaccines, especially as a priming immunization, has been well proved in different HIV vaccine studies, the immunogenicity of DNA vaccines is generally moderate. Novel adjuvant is necessary for improving the immunogenicity of DNA vaccine [73]. Multiple groups have demonstrated the potential of co-administering plasmid DNA that express cytokines, chemokines, or co-stimulatory molecules together with plasmids encoding target viral antigens [74]. The potency of DNA vaccines can be developed by the co-delivery of plasmid-encoded molecular adjuvants. The pDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), hematopoietic factor fms-like tyrosine kinase 3 ligand (Flt-3L), and interleukin-12 (IL-12) could markedly enhance cell-mediated immune responses elicited by an HIV-1 *env* pDNA vaccine in BALB/c mice [74]. Plasmid GM-CSF also increased the immune responses elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pGM-CSF as a vaccine adjuvant appeared to increase antigen-specific proliferative responses and the percentage of polyfunctional memory CD8+ T cells. Co-delivery of pFlt-3L with pGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pGM-CSF with pIL-12 significantly enhanced Env-specific prolifera‐

replication in the absence of antiretroviral therapy) [7].

intervention [72].

120 Advances in Molecular Retrovirology

**6. Adjuvants**

Recombinant adjuvants composed of a fusion between surfactant protein-D (SP-D) and either CD40 ligand (CD40L) or GITR ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. It was demonstrated that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) including 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhance immune responses to an HIV-1 Gag DNA vaccine. Importantly, the SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT constructs enhanced CD8+ T-cell avidity and CD8+ / CD4+ T-cell proliferation 7 weeks after vaccination [81]. Also, the SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF constructs increased Gag-specific IL-2 secretion in memory T cells, suggesting their potency to elevate the number of self-renewing Gag-specific CD8+ and CD4+ T cells. Finally, the SP-D-OX40L and SP-D-CD70 adjuvants augmented IgG2a but not IgG1 antibody responses in the immunized animals. Interestingly, the B-cell-activating protein BAFF did not enhance anti-Gag antibody responses when administered as an SP-D fusion adjuvant, but augmented CD4+ and CD8+ T-cell responses. Indeed, various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid [81]. Several studies indicated that 4-1BB and 4-1BB ligand (4-1BBL) interactions are important for inducing robust CTL responses and also long-lived memory T cells. Recently, plasmid DNAs expressing either the membrane bound or soluble form of 4-1BBL were designed to enhance the Gag DNA vaccine as an adjuvant. The data showed that 4-1BBL DNA increased the Gag-specific IgG and cellular immune responses. Importantly, the expression of Gag and 4-1BBL from the same plasmid was critical for the adjuvant activity [82].

To improve the immunogenicity of DNA vaccines, some studies were focused on the immu‐ noglobulin (Ig) fusion antigen. These reports showed that cytokine-coding plasmids fused with Ig have higher expression efficiency and better adjuvanticity. Furthermore, these plasmids have features that make them useful such as augmentation of half-life *in vivo*, formation of a multivalent antigen, and solubilization of hydrophobic proteins [83]. The possibility of increasing HIV gp120-specific cellular immune responses was determined in mice using a DNA vaccine encoding a mouse Ig fragment fused with gp120 in two directions (gp120-Ig or Ig-gp120). *In vitro* expression analysis revealed that the efficiency of HIV gp120 protein expression was higher in cells transfected with the gp120-Ig-coding plasmid (pGp120Ig) than in those transfected with the gp120 and Ig-gp120 expression plasmids (pGp120 and pIgGp120, respectively). The gp120-Ig-coding plasmid elicited more HIV-specific CD8+ T cells and effector memory CD8+ T cells than pGp120 in immunized mice. Furthermore, pGp120Ig significantly reduced the viral load after challenge with an HIV Env gp160 expressing *vaccinia* virus. These results represented that covalent antigen modification with an Ig sequence can modulate antigen-specific cellular immune responses [83].

Conversely, polysaccharide and nucleic acid fraction extracted from *Mycobacterium bovis bacillus Calmette–Guérin* (BCG-PSN) could be used as a novel adjuvant of DNA vaccine to elicit potent cellular and humoral immune responses against the HIV-1 Env antigen in BALB/c mouse model. In this experiment, the BCG-PSN was mixed with 10 μg or 100 μg of DNA vaccine and injected intramuscularly two or three times. BCG-PSN co-immunization with 10 μg DNA vaccine could elicit cellular and humoral immune responses which were comparable to that induced by 100 μg DNA vaccine alone. Moreover, BCG-PSN could activate TLR signaling pathways and induce Th1-type cytokine secretion. These findings suggested that BCG-PSN can be applied as a new and effective adjuvant for DNA vaccination [73].

Chemokines are largely bioactive inflammatory molecules which play a major role in a variety of immune and inflammatory responses, acting primarily as chemoattractants and activators of various leukocytes. In addition, some chemokines play a critical role in the transmission and progression of HIV-1 and HIV-2 viruses responsible for AIDS. Recent studies have indicated that chemokines and their receptors may play an important role in the differentiation and expansion of T cells in response to immune activation. These regulatory properties of chemokines make them suitable as molecular adjuvants [84]. For example, the modulation and regulation of immune responses were evaluated from the co-delivery of two β-chemokines as gene expression cassettes, MIP-1α or RANTES, along with HIV-1 DNA immunogen constructs. The data showed that MIP-1α had the greatest effect on antibody responses. In addition, coexpression of MIP-1α also modulated the shift of immune responses to Th2-type (*i.e.*, the increase of IgG1/IgG2a ratio). RANTES co-immunization also enhanced the levels of antigenspecific Th1 and CTL responses. The use of chemokine adjuvanted vaccines as HIV vaccine modulators may be important due to the interesting relationship between HIV cell entry and the receptors for β-chemokines. Indeed, β-chemokines as vaccine adjuvants increased βchemokine production in an antigen-specific manner [84].

#### **7. Heterologous prime-boost strategies**

plasmid DNAs expressing either the membrane bound or soluble form of 4-1BBL were designed to enhance the Gag DNA vaccine as an adjuvant. The data showed that 4-1BBL DNA increased the Gag-specific IgG and cellular immune responses. Importantly, the expression of

To improve the immunogenicity of DNA vaccines, some studies were focused on the immu‐ noglobulin (Ig) fusion antigen. These reports showed that cytokine-coding plasmids fused with Ig have higher expression efficiency and better adjuvanticity. Furthermore, these plasmids have features that make them useful such as augmentation of half-life *in vivo*, formation of a multivalent antigen, and solubilization of hydrophobic proteins [83]. The possibility of increasing HIV gp120-specific cellular immune responses was determined in mice using a DNA vaccine encoding a mouse Ig fragment fused with gp120 in two directions (gp120-Ig or Ig-gp120). *In vitro* expression analysis revealed that the efficiency of HIV gp120 protein expression was higher in cells transfected with the gp120-Ig-coding plasmid (pGp120Ig) than in those transfected with the gp120 and Ig-gp120 expression plasmids (pGp120 and pIgGp120, respectively). The gp120-Ig-coding plasmid elicited more HIV-specific

pGp120Ig significantly reduced the viral load after challenge with an HIV Env gp160 expressing *vaccinia* virus. These results represented that covalent antigen modification with

Conversely, polysaccharide and nucleic acid fraction extracted from *Mycobacterium bovis bacillus Calmette–Guérin* (BCG-PSN) could be used as a novel adjuvant of DNA vaccine to elicit potent cellular and humoral immune responses against the HIV-1 Env antigen in BALB/c mouse model. In this experiment, the BCG-PSN was mixed with 10 μg or 100 μg of DNA vaccine and injected intramuscularly two or three times. BCG-PSN co-immunization with 10 μg DNA vaccine could elicit cellular and humoral immune responses which were comparable to that induced by 100 μg DNA vaccine alone. Moreover, BCG-PSN could activate TLR signaling pathways and induce Th1-type cytokine secretion. These findings suggested that

an Ig sequence can modulate antigen-specific cellular immune responses [83].

BCG-PSN can be applied as a new and effective adjuvant for DNA vaccination [73].

Chemokines are largely bioactive inflammatory molecules which play a major role in a variety of immune and inflammatory responses, acting primarily as chemoattractants and activators of various leukocytes. In addition, some chemokines play a critical role in the transmission and progression of HIV-1 and HIV-2 viruses responsible for AIDS. Recent studies have indicated that chemokines and their receptors may play an important role in the differentiation and expansion of T cells in response to immune activation. These regulatory properties of chemokines make them suitable as molecular adjuvants [84]. For example, the modulation and regulation of immune responses were evaluated from the co-delivery of two β-chemokines as gene expression cassettes, MIP-1α or RANTES, along with HIV-1 DNA immunogen constructs. The data showed that MIP-1α had the greatest effect on antibody responses. In addition, coexpression of MIP-1α also modulated the shift of immune responses to Th2-type (*i.e.*, the increase of IgG1/IgG2a ratio). RANTES co-immunization also enhanced the levels of antigenspecific Th1 and CTL responses. The use of chemokine adjuvanted vaccines as HIV vaccine modulators may be important due to the interesting relationship between HIV cell entry and

T cells than pGp120 in immunized mice. Furthermore,

Gag and 4-1BBL from the same plasmid was critical for the adjuvant activity [82].

CD8+

122 Advances in Molecular Retrovirology

T cells and effector memory CD8+

Most of the current DNA vaccines utilize CMV, β-actin, or muscle-specific desmin promoters to potentiate expression of one or two fused genes of HIV-1 including the Env, Gag, Pol, and Tat. DNA vaccines comprising multiple plasmids encoding different HIV-1 proteins have been used to obtain a broader spectrum of immunity than individual plasmids expressing single proteins. The use of these plasmid DNA vaccines proved to be safe and immunogenic in macaques; however, these constructs needed to be boosted with viral proteins expressed by various vector systems including recombinant *pox virus*, modified *vaccinia virus Ankara*, and *adenovirus* for enhancing their efficiency in preventing AIDS [85].

The heterologous prime-boost regimen uses the ability of the immune system to generate large numbers of secondary antigen-specific T cells following an initial priming step. The same antigen is delivered subsequently using different vectors. Following a priming immunization, the antigen-specific T-cell populations develop to modest levels and then reduce. Indeed, a percentage of these cells transform into antigen-specific memory T cells. In a heterologous boost, because the priming and boosting vectors are different, T cells that specifically target the viral vector are not boosted and do not activate cell number control mechanisms, therefore allowing for greater development of the disease antigen-specific T-cell populations [82]. Several groups have now established that heterologous prime-boost regimens are the most potent strategies to induce cellular immune responses [86, 87]. In a plasmid DNA vaccine priming and viral vector-boosting regimen, the order of DNA followed by recombinant virus is important, as the reverse order did not induce higher levels of antigen-specific CD8+ T cells. It seems that the cytokine microenvironment created by a local virus infection during boosting is responsible for the efficient expansion of effector T cells [86]. In 2004, a consecutive immu‐ nization strategy involving priming with DNA and boosting with recombinant fowlpoxvirus (rFPV) vaccines encoding multiple common HIV-1 antigens was evaluated in 30 macaques. The vaccines were well tolerated, and a significant enhancement of DNA-vaccine primed HIV-1-specific T-lymphocyte responses was observed following rFPV boosting. Co-expression of IFNγ or IL-12 by the rFPV vaccines did not further enhance immune responses [88]. In addition, a subtype A or B HIV gp160 plasmid DNA and Env gp140 trimeric glycoprotein coimmunization was superior to immunization with glycoprotein alone by enhancing neutral‐ izing antibodies. These data showed that co-delivering DNA and protein can increase antibody responses to Env. Hence, this approach has the potential to simplify vaccine regimens by inducing higher antibody responses using fewer vaccinations, an advantage for a successful HIV vaccine design [89].

The reports showed that co-immunization of a DNA vaccine encoding HIV-1P24-Nef with GM-CSF in DNA priming and peptide boost strategy increases the immunogenicity of the candidate vaccine. Cytokine profile studies showed that both IL-4 and IFN-γ levels were increased. Also, co-immunization with GM-CSF resulted in a higher level of total IgG, comprising approximately equal levels of both specific IgG1 and IgG2a subtypes. Taken together, the results suggested that GM-CSF is able to induce long-term memory for the HIV-1 P24-Nef vaccine candidate [90]. Recent studies have used DNA/protein or DNA/adeno-vector regimens for HIV immunization. The essential mechanisms of heterogeneous prime/boost regimens are not well understood, but DNA priming results in much lower antigen expression compared to protein vaccines, and this may prime T-helper cell responses with the humoral response subsequently being boosted by the high-dose protein or viral vector (*e.g.*, RV 144 tested in clinical trials) [87].

#### **8. Clinical trials**

Several vaccine candidates were used in different phases of clinical trials. DNA prime-viral vector boost regimens have become the primary choice for stimulation of T-cell immune responses. For example, *Poxvirus* vector-based vaccines including the *Modified Vaccinia Ankara* and the genetically modified NYVAC-based vaccines appeared to be efficient in inducing the immune responses and could be evaluated in combination with DNA priming in clinical trials [91]. In addition, the safety and immunogenicity of several *Canarypox-*based vaccines with multiple HIV-1 gene inserts have been studied in humans. A phase III trial, RV144, using *Canarypox* (vCP1521) prime and AIDSVAX B/E boost has demonstrated modest protective efficacy in Thailand. The protection in RV144 trial was short-time and needed to use the additional boosters in participants for improving recall responses and continuing protection among them. AIDSVAX was also a component of the prime-boost (ALVAC/ AIDSVAX) RV 144 vaccine in Thailand that showed successful results. In both cases, the vaccines targeted gp120 and were specific for the geographical regions. Among the adenoviral vector vaccine candidates, replication-defective Ad5 candidate indicated high immunogenic‐ ity in phase I clinical trials and reduced viral load in the SHIV/NHP model. However, this strategy failed to prevent new infections as well as reduce post-infection viral RNA levels in the vaccinated individuals in phase IIb. Furthermore, participants with preexisting antibodies against Ad5 vector showed increased HIV infection rates [91]. The heterologous prime-boost strategy using DNA prime and Ad5 boost was considered to avoid the problem of preexisting immunity. It has been shown that the preexisting Ad5-neutralizing antibodies did not affect the levels of cell-mediated responses in the DNA/rAd5 prime-boost recipients, as compared to participants who received rAd5 alone. However, in spite of robust immune responses induced by DNA/Ad5 strategy in phase I and phase II trials, the strategy failed to show protection from new infections in phase IIb [91]. Generally, prime-boost vaccination is an efficient approach compared to other strategies, but it still needs to develop against HIV infections in future.

#### **9. Challenges for HIV vaccines**

candidate vaccine. Cytokine profile studies showed that both IL-4 and IFN-γ levels were increased. Also, co-immunization with GM-CSF resulted in a higher level of total IgG, comprising approximately equal levels of both specific IgG1 and IgG2a subtypes. Taken together, the results suggested that GM-CSF is able to induce long-term memory for the HIV-1 P24-Nef vaccine candidate [90]. Recent studies have used DNA/protein or DNA/adeno-vector regimens for HIV immunization. The essential mechanisms of heterogeneous prime/boost regimens are not well understood, but DNA priming results in much lower antigen expression compared to protein vaccines, and this may prime T-helper cell responses with the humoral response subsequently being boosted by the high-dose protein or viral vector (*e.g.*, RV 144

Several vaccine candidates were used in different phases of clinical trials. DNA prime-viral vector boost regimens have become the primary choice for stimulation of T-cell immune responses. For example, *Poxvirus* vector-based vaccines including the *Modified Vaccinia Ankara* and the genetically modified NYVAC-based vaccines appeared to be efficient in inducing the immune responses and could be evaluated in combination with DNA priming in clinical trials [91]. In addition, the safety and immunogenicity of several *Canarypox-*based vaccines with multiple HIV-1 gene inserts have been studied in humans. A phase III trial, RV144, using *Canarypox* (vCP1521) prime and AIDSVAX B/E boost has demonstrated modest protective efficacy in Thailand. The protection in RV144 trial was short-time and needed to use the additional boosters in participants for improving recall responses and continuing protection among them. AIDSVAX was also a component of the prime-boost (ALVAC/ AIDSVAX) RV 144 vaccine in Thailand that showed successful results. In both cases, the vaccines targeted gp120 and were specific for the geographical regions. Among the adenoviral vector vaccine candidates, replication-defective Ad5 candidate indicated high immunogenic‐ ity in phase I clinical trials and reduced viral load in the SHIV/NHP model. However, this strategy failed to prevent new infections as well as reduce post-infection viral RNA levels in the vaccinated individuals in phase IIb. Furthermore, participants with preexisting antibodies against Ad5 vector showed increased HIV infection rates [91]. The heterologous prime-boost strategy using DNA prime and Ad5 boost was considered to avoid the problem of preexisting immunity. It has been shown that the preexisting Ad5-neutralizing antibodies did not affect the levels of cell-mediated responses in the DNA/rAd5 prime-boost recipients, as compared to participants who received rAd5 alone. However, in spite of robust immune responses induced by DNA/Ad5 strategy in phase I and phase II trials, the strategy failed to show protection from new infections in phase IIb [91]. Generally, prime-boost vaccination is an efficient approach compared to other strategies, but it still needs to develop against HIV

tested in clinical trials) [87].

124 Advances in Molecular Retrovirology

**8. Clinical trials**

infections in future.

There are some major challenges against HIV vaccine design as described below. Figure 1 shows these challenges briefly.

**Figure 1.** Challenges for HIV-preventive vaccines

#### **9.1. Designing trimeric HIV-1 envelopes**

The challenge remains to develop HIV-1 immunogens that will elicit protective immunity [13]. The challenge is to design, engineer, and produce a pure stable envelope immunogen that mimics the antigenic profile of the functional envelope spike. The engineered trimeric envelope was unable to induce bNAb in animals. Modification of the trimers, including removal of individual glycans proximal to CD4-binding region, elimination of the glycosylation site near the gp41 loop, linker-stabilized gp140 trimeric envelopes, have resulted in improved immu‐ nogenicity but have not yielded the desired bNAb. A combination of mosaic envelopes increased the magnitude of NAbs but not the breadth of the response in macaques. Therefore, no trimeric envelope induces bNAb in humans [1]. Some studies showed that HIV-1 bNAbs identifies four conserved Env targets for HIV neutralization. To date, more than 30 bNAbs specific for conserved neutralizing Env epitopes have been characterized [17].

#### **9.2. Why does vaccination with HIV envelope not induce bNAbs?**

Whether bNAb will effectively confer protection against HIV infection in humans remains unknown. An alternative to inducing bNAb by vaccination with immunogens is to deliver these bnMAbs with viral vectors (*e.g.*, adeno-associated virus (AAV) gene transfer vector expressing antibodies or antibody-like immunoadhesins). This approach generated a longlasting neutralizing activity in serum of macaques conferring complete protection against intravenous challenge with virulent SIVmac316. Similarly, full protection against intravenous HIV-1 challenge was observed in humanized mice receiving AAV carrying b12, while those receiving AAV carrying 2G12, 4E10, and 2F5 were partially protected [1]. A recent study has demonstrated that up to 50% of HIV-infected individuals will make cross-reactive antibodies that neutralize 50% of HIV primary strains. However, when bNAbs develop in HIV infection, they only occur after 2–4 years of infection. In contrast, no vaccine immunizations to date have induced high levels of bNAbs. The bNAbs are targeted to one of five conserved sites on the HIV Env trimer: the CD4 binding site, the membrane proximal gp41 region, the V3-glycan site, the V1V2-glycan site, and gp41-gp120 bridging regions [51]. Each of these sites is protected by surrounding glycans, and each one of these sites is restricted in access, such that relatively few antibody variable heavy (VHDJH) and variable light (VL) combinations may be used to bind these Env sites. Example of restricted VHDJH/VL usage is the use of VH1-2 paired with a 5 aa VL complementarity-determining region 3 (LCDR3) for the VRC01 type of CD4 binding site bNAb, and the use of VH1-69, Vk3-20 for 4E10-like gp41 bNAbs. Moreover, all bNAbs have one or more unusual features, including high levels of somatic mutations, and poly- or autoreactivity that can result in immune tolerance control for bNAbs. However, in the simianhuman immunodeficiency virus rhesus macaque challenge model, passive infusion of the new bNAbs could potently protect against SHIV challenge [51].

#### **9.3. Effective adjuvants**

Adjuvants are important for the use of recombinant envelope immunogens, since these proteins by themselves generate only weak immune responses. For potent vaccine formula‐ tions delivered by mucosal routes, incorporation of adjuvants that controls the potential of innate immune modulators is important for overcoming immune tolerance and enhancing the immunogenicity of co-administered antigens [25]. The RV144 trial used alum as an adjuvant, which was then the only licensed vaccine adjuvant. However, alum is not believed to support robust cellular immune responses. Also, bacterial toxins are the most potent mucosal adjuvant candidates but concerns remain regarding their safety even when mutated to reduce toxicity. In contrast, ligands for TLRs 7/8 and 9 serve as potent adjuvants for parenteral and mucosal vaccines based on plasmid DNA, viral vectors, and recombinant proteins [25]. In particular, CpG-containing synthetic oligodeoxynucleotides (CpG-ODN) that activate TLR9 on dendritic cells appear potent in stimulating antigen presentation and induction of antigen-specific immune responses. The synthetic glycolipid α-galactosylceramide has been tested primarily in cancer immunotherapy studies because of its capacity to serve as a ligand and potent activator of invariant natural killer T cells. In addition, the repeated mucosal delivery of α-GalCer adjuvant was done in primary and booster immunizations that resulted in repeated activation of NKT cells and DC to progressively increase adaptive immune responses [25]. The parallel development of adjuvants along with better HIV-1 immunogens will be needed for a successful AIDS vaccine. Additional comparative testing will be required to determine the optimal adjuvant and immunogen regimen that can elicit antibody responses capable of blocking HIV-1 transmission [92].

Both flagellin (fliC) and IL-18 (interferon-γ-inducing factor) have been developed as adjuvants to improve immunogenicity in DNA-vaccinated hosts. An HIV-1 Gag plasmid encodes a protein harboring broad epitopes for CTLs [93]. The immunogenicity of BALB/c mice immu‐ nized with an HIV-1 Gag plasmid (pVAX/Gag), combined with a chimeric plasmid encoding IL-18 fused to flagellin (pcDNA3/IL-18\_fliC) or a single plasmid encoding IL-18 (pcDNA3/ IL-18) and/or flagellin (pcDNA3/fliC), was studied. The IL-18 and flagellin fusion protein effectively induced IFN-γ by lymphocytes. During a 12-week immunization, both Gag-specific IgG in sera and spleen cell proliferation were elevated in all murine groups. However, the IgG2a/IgG1 ratio, Th1 cytokine (IL-2 and IFN-γ) production, and the proportion of Gagspecific CD3+ CD8+ IFN-γ-secreting cells were significantly increased in the murine group coimmunized with the pVAX/Gag plasmid and pcDNA3/IL-18\_fliC compared with the mice immunized with the pVAX/Gag plasmid combined with either the pcDNA3/fliC or pcDNA3/ IL-18 plasmid or both of them. The data suggested that the chimeric plasmid encoding IL-18 fused to flagellin can be used as an adjuvant-like plasmid to improve the Th1 immune response, particularly for the induction of CD3+ CD8+ IFN-γ-secreting cells in Gag plasmidvaccinated mice [93].

#### **9.4. High mutation rate of HIV-1**

The high mutation rate of HIV-1 and tolerance for genetic diversity represented central challenges for vaccine design. Because the immune response is itself adaptive, the optimal HIV-1 sequence within an individual also differs over time. HIV-1 develops specific mutations within its genome that allow it to escape detection by human leukocyte antigen (HLA) class I-restricted immune responses, notably those of CD8+ CTLs. HLA thus represents a major force driving the evolution and diversity of HIV-1 within individuals [94]*.*

#### **9.5. Escaped variants**

identifies four conserved Env targets for HIV neutralization. To date, more than 30 bNAbs

Whether bNAb will effectively confer protection against HIV infection in humans remains unknown. An alternative to inducing bNAb by vaccination with immunogens is to deliver these bnMAbs with viral vectors (*e.g.*, adeno-associated virus (AAV) gene transfer vector expressing antibodies or antibody-like immunoadhesins). This approach generated a longlasting neutralizing activity in serum of macaques conferring complete protection against intravenous challenge with virulent SIVmac316. Similarly, full protection against intravenous HIV-1 challenge was observed in humanized mice receiving AAV carrying b12, while those receiving AAV carrying 2G12, 4E10, and 2F5 were partially protected [1]. A recent study has demonstrated that up to 50% of HIV-infected individuals will make cross-reactive antibodies that neutralize 50% of HIV primary strains. However, when bNAbs develop in HIV infection, they only occur after 2–4 years of infection. In contrast, no vaccine immunizations to date have induced high levels of bNAbs. The bNAbs are targeted to one of five conserved sites on the HIV Env trimer: the CD4 binding site, the membrane proximal gp41 region, the V3-glycan site, the V1V2-glycan site, and gp41-gp120 bridging regions [51]. Each of these sites is protected by surrounding glycans, and each one of these sites is restricted in access, such that relatively few antibody variable heavy (VHDJH) and variable light (VL) combinations may be used to bind these Env sites. Example of restricted VHDJH/VL usage is the use of VH1-2 paired with a 5 aa VL complementarity-determining region 3 (LCDR3) for the VRC01 type of CD4 binding site bNAb, and the use of VH1-69, Vk3-20 for 4E10-like gp41 bNAbs. Moreover, all bNAbs have one or more unusual features, including high levels of somatic mutations, and poly- or autoreactivity that can result in immune tolerance control for bNAbs. However, in the simianhuman immunodeficiency virus rhesus macaque challenge model, passive infusion of the new

Adjuvants are important for the use of recombinant envelope immunogens, since these proteins by themselves generate only weak immune responses. For potent vaccine formula‐ tions delivered by mucosal routes, incorporation of adjuvants that controls the potential of innate immune modulators is important for overcoming immune tolerance and enhancing the immunogenicity of co-administered antigens [25]. The RV144 trial used alum as an adjuvant, which was then the only licensed vaccine adjuvant. However, alum is not believed to support robust cellular immune responses. Also, bacterial toxins are the most potent mucosal adjuvant candidates but concerns remain regarding their safety even when mutated to reduce toxicity. In contrast, ligands for TLRs 7/8 and 9 serve as potent adjuvants for parenteral and mucosal vaccines based on plasmid DNA, viral vectors, and recombinant proteins [25]. In particular, CpG-containing synthetic oligodeoxynucleotides (CpG-ODN) that activate TLR9 on dendritic cells appear potent in stimulating antigen presentation and induction of antigen-specific

specific for conserved neutralizing Env epitopes have been characterized [17].

**9.2. Why does vaccination with HIV envelope not induce bNAbs?**

bNAbs could potently protect against SHIV challenge [51].

**9.3. Effective adjuvants**

126 Advances in Molecular Retrovirology

A major challenge is how to induce effective immune responses against escaped variants. It is important that a CTL-based vaccine stimulate effective cellular responses across the range of HLA class I alleles expressed in a host population. These observations have led to the idea that immune-mediated control of HIV-1 replication to levels that slow disease progression might be feasible through the design of vaccines that focus CTL responses against viral regions where escape cannot occur [94]. To date, adenovirus vector prime and pox vector boost vaccines have been one among the most immunogenic vaccines for inducing HIV CD8 T-cell responses in humans. Efforts continue to overcome HIV diversity for T-cell epitope recognition by the *in silico* design of centralized consensus or mosaic HIV gene inserts based on optimizing the coverage of T-cell epitopes in HIV strains in the Los Alamos HIV Sequence Database, or based on conserved epitopes in the vaccine [51].

#### **9.6. Expression of the bNAbs is limited by host tolerance mechanisms**

The studies showed that two human recombinant bNAbs, called 2F5 and 4E10, that bind near the virion membrane to Env gp41 were reactive in human autoantibody assays. In a subsequent study, 2F5 was shown to avidly bind the human protein kynureninase (KYNU), and 4E10 was shown to react with the mammalian RNA splicing factor 3B3. The nominal gp41 epitope of the 2F5 bNAb is the linear peptide ELDKWAS and an identical 6-residue sequence is present in KYNU (ELDKWA). This ELDKWA motif in KYNU is conserved in nearly all mammalian species and absent in all proteins other than the HIV Env. Thus, the autoantigens for these two bNAbs, 2F5 and 4E10, have been identified, suggesting that expression of these bNAbs is limited by host tolerance mechanisms [17].

#### **9.7. Challenges for developing vaccines targeting viral glycan epitopes**

Generation of antibodies to glycans has several challenges: a) due to the inherent weakness of carbohydrate–protein interactions, binding affinities must be enhanced through avidity effects. For example, lectins are able to overcome this problem using interaction of multiple carbohydrate binding domains with arrays of glycan ligands; b) glycoproteins usually always exist as a number of different glycoforms where the same protein backbone is glycosylated with different glycan structures. This microheterogeneity weakens the antigenic response to the individual glycan structures. These multiple conformations may be presented to the immune system further weakening the response; c) glycosylation is ubiquitous to all mam‐ malian cells, thus the host may display tolerance toward these sugars. Generally, these effects result in glycans are poorly immunogenic. The major concern of generating antibodies against self-glycan structures is their potential autoreactivity *in vivo* [95].

#### **9.8. Animal model for preclinical studies**

There are few nonhuman primate models of enhanced HIV susceptibility [96]. Animal model research during the past years has focused on the development of models in order to explore key questions about HIV entry, immune control, and persistence and also their use for testing therapeutic vaccines [97].

#### **9.9. Design of new envelope immunogens**

A major challenge for HIV-1 B-cell vaccine development is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and inter‐ mediate memory B cells associated with the maturation of a broadly neutralizing antibody response. The identification of delivery systems, prime-boost strategies, and synergistic adjuvant combinations is important to induce the magnitude and quality of antigen-specific T-follicular helper cell responses needed to induce somatic hypermutation (SHM) and B-cell maturation against heterologous primary virus envelopes [98].

#### **9.10. Safety of vaccines**

been one among the most immunogenic vaccines for inducing HIV CD8 T-cell responses in humans. Efforts continue to overcome HIV diversity for T-cell epitope recognition by the *in silico* design of centralized consensus or mosaic HIV gene inserts based on optimizing the coverage of T-cell epitopes in HIV strains in the Los Alamos HIV Sequence Database, or based

The studies showed that two human recombinant bNAbs, called 2F5 and 4E10, that bind near the virion membrane to Env gp41 were reactive in human autoantibody assays. In a subsequent study, 2F5 was shown to avidly bind the human protein kynureninase (KYNU), and 4E10 was shown to react with the mammalian RNA splicing factor 3B3. The nominal gp41 epitope of the 2F5 bNAb is the linear peptide ELDKWAS and an identical 6-residue sequence is present in KYNU (ELDKWA). This ELDKWA motif in KYNU is conserved in nearly all mammalian species and absent in all proteins other than the HIV Env. Thus, the autoantigens for these two bNAbs, 2F5 and 4E10, have been identified, suggesting that expression of these bNAbs is

Generation of antibodies to glycans has several challenges: a) due to the inherent weakness of carbohydrate–protein interactions, binding affinities must be enhanced through avidity effects. For example, lectins are able to overcome this problem using interaction of multiple carbohydrate binding domains with arrays of glycan ligands; b) glycoproteins usually always exist as a number of different glycoforms where the same protein backbone is glycosylated with different glycan structures. This microheterogeneity weakens the antigenic response to the individual glycan structures. These multiple conformations may be presented to the immune system further weakening the response; c) glycosylation is ubiquitous to all mam‐ malian cells, thus the host may display tolerance toward these sugars. Generally, these effects result in glycans are poorly immunogenic. The major concern of generating antibodies against

There are few nonhuman primate models of enhanced HIV susceptibility [96]. Animal model research during the past years has focused on the development of models in order to explore key questions about HIV entry, immune control, and persistence and also their use for testing

A major challenge for HIV-1 B-cell vaccine development is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and inter‐ mediate memory B cells associated with the maturation of a broadly neutralizing antibody response. The identification of delivery systems, prime-boost strategies, and synergistic

**9.6. Expression of the bNAbs is limited by host tolerance mechanisms**

**9.7. Challenges for developing vaccines targeting viral glycan epitopes**

self-glycan structures is their potential autoreactivity *in vivo* [95].

on conserved epitopes in the vaccine [51].

128 Advances in Molecular Retrovirology

limited by host tolerance mechanisms [17].

**9.8. Animal model for preclinical studies**

**9.9. Design of new envelope immunogens**

therapeutic vaccines [97].

Safety of vaccines is one of the most important subjects for the design of vaccines that should be determined in clinical trials [99].

#### **9.11. Accessibility of the glycoconjugate vaccines**

Accessibility of these glycoconjugate vaccines in resource-poor regions which bear the highest disease burden from these pathogens remains challenging largely due to high vaccine pricing [100].

#### **9.12. Induction of potent and broadly cross-reactive neutralizing antibody responses**

Induction of potent and broadly cross-reactive neutralizing antibody responses remains a major challenge for the development of HIV vaccines because of the high diversity of gp120. The high glycosylation, large conformational changes, and steric restriction of the epitopes in gp120 during receptor binding and membrane fusion processes prevent the access of antibod‐ ies to these sites [101].

#### **9.13. Challenges associated with antigen immunogenicity**

The failure to date of Env-based antigens to stimulate bNAb is likely to result from several specific reasons that influence BCR recognition of unusual structural antigenic elements:


Preparation of such epitopes will require powerful synthetic chemistry related to scaf‐ folded peptide design. Both MPER bNmAbs 2F5 and 4E10 require a lipid component to their epitopes and up to now this has not been incorporated into a successful immunogen [5].


Finally, the key difficulty in the development of an HIV vaccine is our ignorance of the immune responses that control viral replication, how these responses can be elicited, and how they can be monitored [2]. The question of whether to focus on induction of antibody or CTLs continues to be discussed in the HIV-1 field. However, evidence from many other vaccine-preventable infectious diseases indicates that antibody titers correlate with protection from infection, but CTL-mediated immune responses are required for protection against disease. This suggests that a dual approach is still necessary. Aspects of CTL vaccine technology such as replicating or persistent vectors may need to express Env-based antigens to allow long-term antigenic exposure for the induction of bNAb. In contrast, approaches to elicit bNmAbs may need to be immunologically compatible with the generation of a parallel CTL response.

#### **9.14. Conclusions**

The development of a safe and effective vaccine for HIV is a major global priority. To date, efforts to design an HIV vaccine have not been successful due to HIV diversity, HIV integration into the host genome, and ability of HIV to consistently evade antiviral immune responses. While the RV144 immunization strategy remains a priority for future efficacy trials, newer prime-boost mosaic and conserved sequence immunization strategies inducing efficient and long-time immune responses as well as the development of immunogens inducing broadly neutralizing antibodies should be followed and tested in humans. Recent success in isolation of potent broadly neutralizing antibodies, in discovery of mechanisms of bNAb induction and atypical mechanisms of CD8 T-cell killing of HIV infected cells, has opened new ways for HIV vaccine design. Indeed, the most protective HIV vaccine will require the combination of T-cellinducing and antibody-inducing vaccine candidates with appropriate adjuvant formulations, since the innate and adaptive arms of the immune system cooperate for virus neutralization and pathogen-infected cell elimination. In general, acceleration of vaccine discovery depends on basic research and new technologies. Novel strategies should be safe, but rapidly tested in humans.

#### **10. Key points**

Preparation of such epitopes will require powerful synthetic chemistry related to scaf‐ folded peptide design. Both MPER bNmAbs 2F5 and 4E10 require a lipid component to their epitopes and up to now this has not been incorporated into a successful immunogen

**e. Germline BCR recognition and requirement for extensive antibody affinity matura‐ tion**: There are two possible consequences of the steric constraints imposed on BCRs during the recognition of structurally unusual antigens: a) the frequency of germline BCRs available to recognize such complex antigens will be low, thus an extensive degree of affinity maturation will be required to generate a high-affinity bNAbs recognizing structurally "difficult" epitopes; b) the germline BCR affinity for a bNmAb epitope may be undetectable. A feasible outcome of these constraints is that the host will require longterm antigen exposure to select and clonally expand the rare B cells with appropriate BCRs and to affinity mature them into bNAbs, because most bNmAbs arise in individuals after

**f. Conceptual concerns relating to epitope recognition by BCRs:** There are concerns that isolating an epitope from its antigenic context will not lead to re-elicitation of the same type of antibody against the epitope, which is a reasonable concern. However, although an epitope mimic may not re-stimulate an antibody identical to the template bNmAb, it may be sufficiently balance between elicited Ab and epitope mimic to allow specific binding to trimeric Env. If this is achieved, trimeric Env may be used to boost and affinity

**g. Responders and non-responders**: The finding that among large cohorts of HIV-1-infected individuals only a minor percentage makes a bNmAb response suggests that this may

Finally, the key difficulty in the development of an HIV vaccine is our ignorance of the immune responses that control viral replication, how these responses can be elicited, and how they can be monitored [2]. The question of whether to focus on induction of antibody or CTLs continues to be discussed in the HIV-1 field. However, evidence from many other vaccine-preventable infectious diseases indicates that antibody titers correlate with protection from infection, but CTL-mediated immune responses are required for protection against disease. This suggests that a dual approach is still necessary. Aspects of CTL vaccine technology such as replicating or persistent vectors may need to express Env-based antigens to allow long-term antigenic exposure for the induction of bNAb. In contrast, approaches to elicit bNmAbs may need to be

The development of a safe and effective vaccine for HIV is a major global priority. To date, efforts to design an HIV vaccine have not been successful due to HIV diversity, HIV integration into the host genome, and ability of HIV to consistently evade antiviral immune responses. While the RV144 immunization strategy remains a priority for future efficacy trials, newer

mature those B cells reactive with the epitope mimetic [5].

immunologically compatible with the generation of a parallel CTL response.

apply also to responses to vaccination [5].

[5].

130 Advances in Molecular Retrovirology

chronic HIV-1 infection [5].

**9.14. Conclusions**


#### **Author details**

#### Azam Bolhassani\*

Address all correspondence to: A\_bolhasani@pasteur.ac.ir; azam.bolhassani@yahoo.com

Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran

#### **References**


[12] Excler, J.L., Robb, M.L., Kim, J.H. (2014). HIV-1 vaccines: challenges and new per‐ spectives. Hum. Vaccin. Immunother. 10(6), 1734–1746

**Author details**

132 Advances in Molecular Retrovirology

Azam Bolhassani\*

**References**

cine. Vaccine, 1–9

munother., 11(4),1022–1029

Vaccine, 33, 1243–1249

28(12), 1701–1718

Clin. North. Am., 28(4), 615–631

and tribulations. Immunol. Res. 60(1), 35–37

Address all correspondence to: A\_bolhasani@pasteur.ac.ir; azam.bolhassani@yahoo.com

[1] Excler, J.L., Robb, M.L., Kim, J.H. (2015). Prospects for a globally effective HIV-1 vac‐

[2] de Goede, A.L., Vulto, A.G., Osterhaus, A.D., Gruters, R.A. (2015). Understanding HIV infection for the design of a therapeutic vaccine; Part I: Epidemiology and

[3] Richert, L., Lhomme, E., Fagard, C., Lévy, Y., Chêne, G., Thiébaut, R. (2015). Recent developments in clinical trial designs for HIV vaccine research. Hum. Vaccin. Im‐

[4] Wilson, C.B., Karp, C.L. (2015). Can immunological principles and cross-disciplinary science illuminate the path to vaccines for HIV and other global health challenges?

[5] Schiffner, T., Sattentau, Q.J., Dorrell, L. (2013). Development of prophylactic vaccines

[6] VISR Working Group of the Global HIV Vaccine Enterprise. (2015). HIV vaccine-in‐ duced sero-reactivity: A challenge for trial participants, researchers, and physicians.

[7] Mylvaganam, G.H., Silvestri, G., Amara, R.R. (2015). HIV therapeutic vaccines: mov‐

[8] Goepfert, P., Bansal, A. (2014). Human immunodeficiency virus vaccines. Infect. Dis.

[9] Hanke, T. (2014). Conserved immunogens in prime-boost strategies for the next-gen‐

[10] Alchin, D.R. (2014). HIV vaccine development: an exploratory review of the trials

[11] Pavot, V., Rochereau, N., Lawrence, P., Girard, M.P., Genin, C., Verrier, B., Paul, S. (2014). Recent progress in HIV vaccines inducing mucosal immune responses. AIDS,

Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran

pathogenesis of HIV infection. Ann. Pharm. Fr., 73(2), 87–99

ing towards a functional cure. Curr. Opin. Immunol., 35, 1–8

eration HIV-1 vaccines. Expert Opin. Biol. Ther.,14(5), 601–616

Philos. Trans. R. Soc. Lond. B. Biol. Sci.,19, 370

against HIV-1. Retrovirology, 10(72), 1–16


[40] Vaccari, M., Gordon, S.N., Fourati, S., Schifanella, L., Cameron, M., Keele, B.F., et al. (2014). Adjuvant dependent mucosal V2 responses and RAS activation in vaccine in‐ duced protection from SIVmac251 acquisition. In: OA25.01. HIV R4P.

[26] Zolla-Pazner, S. (2014). A critical question for HIV vaccine development: which anti‐

[28] Gottardo, R., Bailer, R.T., Korber, B.T., Gnanakaran, S., Phillips, J., Shen, X., et al. (2013). Plasma IgG to linear epitopes in the V2 and V3 regions of HIV-1 gp120 corre‐ late with a reduced risk of infection in the RV144 vaccine efficacy trial. PLoS One, 8,

[29] Yates, N.L., Liao, H.X., Fong, Y, Decamp, A., Vandergrift, N.A., Williams, W.T., et al. (2014). Vaccine-induced Env V1–V2 IgG3 correlates with lower HIV-1 infection risk

[30] Chung, A.W., Ghebremichael, M., Robinson, H., Brown, E., Choi, I., Lane, S., et al. (2014). Poly-functional Fc-effector profiles mediated by IgG subclass selection distin‐

[31] O'Connell, R.J., Kim, J.H., Excler, J.L. (2014). The HIV-1 gp120 V1V2 loop: structure, function and importance for vaccine development. Expert Rev. Vaccines, 1–12

[32] Plotkin, S.A., Gilbert, P.B. (2012). Nomenclature for immune correlates of protection

[33] Karasavvas, N., Karnasuta, C., Ngauy, V., Vasan, S., Tricharavoj, R., de Souza, M.S., et al. (2013). Investigation of antibody responses induced in RV305 a late boost vacci‐ nation of HIV-1 uninfected volunteers that participated in RV144, a Thai trial. In:

[34] Akapirat, S., Karnasuta, C., Madnote, S., Savadsuk, H., Puangkaew, J., Rittiroongrad, S., et al. (2014). HIV-specific antibody in rectal secretions following late boosts in

[35] Su, B., Moog, C. (2014). Which antibody functions are important for an HIV vaccine?

[36] Kohler, H. (2015). Novel vaccine concept based on back-boost effect in viral infection.

[37] O'Hagan, D.T., Ott, G.S., De Gregorio, E., Seubert, A. (2012). The mechanism of ac‐ tion of MF59-an innately attractive adjuvant formulation. Vaccine, 30, 4341–4348

[38] Leroux-Roels, I., Koutsoukos, M., Clement, F., Steyaert, S., Janssens, M., Bour‐ guignon, P., et al. (2010). Strong and persistent CD4+ T-cell response in healthy adults immunized with a candidate HIV-1 vaccine containing gp120, Nef and Tat an‐

[39] Garcon, N., Van Mechelen, M. (2011). Recent clinical experience with vaccines using MPL- and QS-21-containing adjuvant systems. Expert Rev. Vaccines, 10, 471–486

tigens formulated in three adjuvant systems. Vaccine, 28, 7016–7024

[27] Mascola, J.R. (2007). HIV/AIDS: allied responses. Nature, 449, 29–30

and declines soon after vaccination. Sci. Transl. Med., 6, 228–239

after vaccination. Clin. Infect. Dis., 54, 1615–1617

RV144 participants (RV305). In: OA11.05, HIV R4P.

Frontiers in Immunology, HIV and AIDS, 5, 1–12

P03.68LB, AIDS Vaccine

Vaccine, 33, 3274–3275

guish RV144 and VAX003 vaccines. Sci. Transl. Med., 6(228), 228–238

bodies to induce? Science, 345, 167–168

e75665

134 Advances in Molecular Retrovirology


affect disease progression in HIV-1-infected male subjects: results from a randomized placebo-controlled trial (the step study). J. Infect. Dis., 203,765–772


[64] Bolhassani, A., Rafati, S. (2013). Mini-chaperones: potential immuno-stimulators in vaccine design. Hum. Vaccin. Immunother., 9, 153–161

affect disease progression in HIV-1-infected male subjects: results from a randomized

[53] Li, F., Finnefrock, A.C., Dubey, S.A., Korber, B.T., Szinger, J., Cole, S., et al. (2011). Mapping HIV-1 vaccine induced T-cell responses: bias towards less-conserved re‐ gions and potential impact on vaccine efficacy in the step study. PLOS ONE, 6,

[54] Ratto-Kim, S., Currier, J.R., Cox, J.H., Excler, J.L., Valencia-Micolta, A., Thelian, D., et al. (2012). Heterologous prime-boost regimens using rAd35 and rMVA vectors elicit stronger cellular immune responses to HIV proteins than homologous regimens.

[55] Santra, S., Liao, H.X., Zhang, R., Muldoon, M., Watson, S., Fischer, W., et al. (2010). Mosaic vaccines elicit CD8+ T lymphocyte responses that confer enhanced immune

[56] Barouch, D.H., O'Brien, K.L., Simmons, N.L., King, S.L., Abbink, P., Maxfield, L.F., et al. (2010). Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune

[57] Kulkarni, V., Valentin, A., Rosati, M., Rolland, M., Mullins, J.I., Pavlakis, G.N., et al. (2014). HIV-1 conserved elements p24CE DNA vaccine induces humoral immune re‐

[58] Stephenson, K.E., SanMiguel, A., Simmons, N.L., Smith, K., Lewis, M.G., Szinger, J.J., et al. (2012). Full-length HIV-1 immunogens induce greater magnitude and compara‐ ble breadth of T lymphocyte responses to conserved HIV-1 regions compared with conserved-region-only HIV-1 immunogens in rhesus monkeys. J. Virol. 86, 11434–

[59] Hansen, S.G., Ford, J.C., Lewis, M.S., Ventura, A.B., Hughes, C.M., Coyne-Johnson, L., et al. (2011). Profound early control of highly pathogenic SIV by an effector mem‐

[60] Hansen, S.G., Piatak, J. M., Ventura, A.B., Hughes, C.M., Gilbride, R.M., Ford, J.C., et al. (2013). Immune clearance of highly pathogenic SIV infection. Nature, 50, 100–104

[61] Bolhassani, A., Yazdi, S.R. (2009). DNA immunization as an efficient strategy for vac‐

[62] Habibzadeh, N., Bolhassani, A., Vahabpour, R., Sadat, S.M. (2015). How can Improve DNA Vaccine Modalities as a Therapeutic Approach against HIV Infections? J. AIDS

[63] Daemi, A., Bolhassani, A., Rafati, S., Zahedifard, F., Hosseinzadeh, S., et al. (2012). Different domains of glycoprotein 96 influence HPV16 E7 DNA vaccine potency via electroporation mediated delivery in tumor mice model. Immunol. Lett., 148, 117–125

sponses with broad epitope recognition in macaques. PLOS ONE, 9, e111085

coverage of diverse HIV strains in monkeys. Nat. Med., 16, 324–328

responses in rhesus monkeys. Nat. Med., 16, 319–323

ory T-cell vaccine. Nature, 473, 523–527

cination. J. Med. Biotechnol., 1, 71–88

Clin. Res., 6(4), 1–8

placebo-controlled trial (the step study). J. Infect. Dis., 203,765–772

e20479

136 Advances in Molecular Retrovirology

11440

PLOS ONE, 7, e45840


HIV-1 using GM-CSF in DNA prime/peptide boost strategy: GM-CSF induced longlived memory responses. Immunol. Lett., 140, 14–20

[91] Shete, A., Thakar, M., Mehendale, S., Paranjape, R. (2014). Is prime boost strategy a promising approach in HIV vaccine development? J. AIDS Clin. Res., 5, 293

[77] Xin, K.Q., Hamajima, K., Sasaki, S., Tsuji, T., Watabe, S., et al. (1999). IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine. Vac‐

[78] Jiang, W., Jin, N., Cui, S., Li, Z., Zhang, L., et al. (2006). Enhancing immune responses against HIV-1 DNA vaccine by coinoculating IL-6 expression vector. J. Virol. Meth‐

[79] Nimal, S., Heath, A.W., Thomas, M.S. (2006). Enhancement of immune responses to an HIV gp120 DNA vaccine by fusion to TNF alpha cDNA. Vaccine, 24, 3298–3308 [80] Huang, Y., Chen, A., Li, X., Chen, Z., Zhang, W., et al. (2008). Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand, alpha-galactosylceramide.

[81] Kanagavelu, S.K., Snarsky, V., Termini, J.M., Gupta, S., Barzee, S., et al. (2012). Solu‐ ble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a

[82] Scott, J.K., Gulzar, N., Klaric, K., Ross, T.M., Lu, S., et al. (2012). DNA vaccines that express the MPER of HIV-1gp41 elicit different antibodies depending upon their

[83] Shimada, M., Yoshizaki, S., Jounai, N., Kondo, A., Ichino, M., et al. (2010). DNA vac‐ cine expressing HIV-1 gp120/immunoglobulin fusion protein enhances cellular im‐

[84] Boyer, J.D., Kim, J., Ugen, K., Cohen, A.D., Ahn, L., et al. (1999). HIV-1 DNA vaccines

[85] Liu, Z., Singh, D.K., Sheffer, D., Smith, M.S., Dhillon, S., et al. (2006). Immunopro‐ phylaxis against AIDS in macaques with a lentiviral DNA vaccine. Virology, 351,

[86] Ivanoff, L.A., Dubay, J.W., Morris, J.F., Roberts, S.J., Gutshall, L., et al. (1992). V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity. Virology,

[87] Li, L., Saade, F., Petrovsky, N. (2012). The future of human DNA vaccines. J. Biotech‐

[88] Shinoda, K., Xin, K.Q., Kojima, Y., Saha, S., Okuda, K., et al. (2006). Robust HIV-spe‐ cific immune responses were induced by DNA vaccine prime followed by attenuated

[90] Mahdavi, M., Ebtekar, M., Khorram Khorshid, H.R., Azadmanesh, K., Hartoonian, C., Hassan, Z.M., (2011). ELISPOT analysis of a new CTL based DNA vaccine for

recombinant vaccinia virus (LC16m8 strain) boost. Clin. Immunol., 119, 32–37 [89] Pissani, F., Malherbe, D.C., Schuman, J.T., Robins, H., Park, B.S., et al. (2014). Im‐ provement of antibody responses by HIV envelope DNA and protein co-immuniza‐

transmembrane and cytoplasmic domains. Retrovirology, 9, P348

cine, 17, 858–866

138 Advances in Molecular Retrovirology

ods, 136, 1–7

Vaccine, 26, 1807–1816

munity. Vaccine 28, 4920–4927

444–454

187, 423–432

nol., 162, 171–182

tion. Vaccine, 32, 507–513

and chemokines. Vaccine, 17, S53–S64

HIV-1 Gag DNA vaccine. Vaccine, 30, 691–702


**Retroviruses as Vectors in Gene Therapy**

### **Retroviral Vectors in Gene Therapy**

Miroslava Matuskova and Erika Durinikova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61844

#### **Abstract**

Several decades ago, the first retroviral vectors were constructed. They have been proved as delivery vehicles in basic and translational research; many of them were used in clini‐ cal trials in the treatment of genetic and immunologic disorders or malignancies to deliv‐ er therapeutic genes into target tissue. Gammaretroviral and lentiviral vectors are popular viral delivery vehicles; their ability to integrate into genome of the host cell ena‐ bles permanent genetic modification of the target cell and long-term expression of the transgene. Besides classical cancer gene therapy, they are used in cell-mediated cancer gene therapy in combination with mesenchymal stromal cells (MSC) or neural progeni‐ tors. Based on the promising preclinical studies, clinical trials with genetically engineered cell vehicles were initiated.

**Keywords:** Retroviral vector, lentiviral vector, gene therapy

#### **1. Introduction**

Besides negative and pathogenic attributes, viruses can also be beneficial when used as delivery vehicles in gene therapy. The advocates of viral vectors even claim that just viruses are the right tools for delivery of foreign genetic information into the cell because they have been evolving for this purpose for millions of years. Gene therapy can be defined as the delivery of nucleic acid into the cell for the purpose of acquiring new features or restoration of phys‐ iologic status. The idea that disorders can be treated by genes arose in the 1960s, when the mechanism of cell transformation by SV40 virus and papovaviruses was described [1]. Gene therapy enables modification of cell by the replacement of non-functional or missing gene, suppression of another gene, or induction of cell death as in the case of oncologic diseases. Monogenic diseases and age-related disorders can be treated by retrovirus-mediated gene therapy, but (retro)viral vectors are most frequently used in cancer gene therapy.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1.1. Basic terminology**

	- **◦** Transient DNA is not integrated into the genome of host cell, genetic modification is temporary.
	- **◦** Stable transgene is an integral part of the genome of the host cell, and it is transferred into daughter cells by cell division.

#### **2. History of gene therapy and retroviral vectors**

The transfer of genetic information among bacteria by a bacteriophage was first described by Joshua Lederberg and Norton Zinder. They named this phenomenon as 'transduction' [2].

The work of Howard M. Temin performed on Rous sarcoma virus (RSV) is the fundamental part in the research of retroviruses and retroviral vectors. He discovered that specific genetic mutations could be inherited as a result of viral infection. Moreover, his study showed that RSV infection required cellular DNA for replication, and genetic information can also flow in the direction RNA → DNA, and he postulated the provirus hypothesis [3-6].

In the 1970s, specific viral genes involved in the transformation were discovered. The SRC and other (proto-)oncogenes with cellular origin were described. The insertional mutagenesis was revealed as another mechanism of transformation [7].

While pioneer work was performed on avian-infectious alpharetroviral Rous sarcoma virus, Moloney murine leukaemia virus (MoMLV) belonging to gammaretroviruses was initially used for the preparation of therapeutic vector [8, 9], and until now, MoMLV-derived constructs along with human immunodeficiency virus (HIV)-derived vectors are most

frequently used. The construction of mutant Moloney murine leukaemia virus defective in the packaging of genomic RNA into virions represents an important step towards the development of retroviral vectors [7]. The first gene delivery systems based on HIV-1 were prepared in the early 1990s [10].

**1.1. Basic terminology**

144 Advances in Molecular Retrovirology

temporary.

genetic information into the target cell.

into daughter cells by cell division.

independently from the genome of the host cell.

able to replicate in target transduced cells.

**2. History of gene therapy and retroviral vectors**

revealed as another mechanism of transformation [7].

packaged in the viral particle).

infects surrounding cells.

**•** Viral vector – a synthetic construct containing given viral sequences determined to transfer

**•** Transduction – transfer of genetic information by a viral vector (genetic information is

**◦** Transient – DNA is not integrated into the genome of host cell, genetic modification is

**◦** Stable – transgene is an integral part of the genome of the host cell, and it is transferred

**•** Episome – foreign genetic information in the cytoplasm or nucleus, which replicates

**•** Replication-competent vector – the genetic information of the virus is complete, and the realisation of the whole life cycle of the virus is facilitated in the target cells. Viral progeny

**•** Replication-defective vector – some viral sequences are removed from the genome; helper cell lines are necessary for the production of virion. Replication-defective vectors are not

The transfer of genetic information among bacteria by a bacteriophage was first described by Joshua Lederberg and Norton Zinder. They named this phenomenon as 'transduction' [2].

The work of Howard M. Temin performed on Rous sarcoma virus (RSV) is the fundamental part in the research of retroviruses and retroviral vectors. He discovered that specific genetic mutations could be inherited as a result of viral infection. Moreover, his study showed that RSV infection required cellular DNA for replication, and genetic information can also flow in

In the 1970s, specific viral genes involved in the transformation were discovered. The SRC and other (proto-)oncogenes with cellular origin were described. The insertional mutagenesis was

While pioneer work was performed on avian-infectious alpharetroviral Rous sarcoma virus, Moloney murine leukaemia virus (MoMLV) belonging to gammaretroviruses was initially used for the preparation of therapeutic vector [8, 9], and until now, MoMLV-derived constructs along with human immunodeficiency virus (HIV)-derived vectors are most

the direction RNA → DNA, and he postulated the provirus hypothesis [3-6].

**•** Transfection – transfer of genetic information by a non-viral system.

**•** Provirus – viral genome integrated into chromosome of the host cell.

The first officially approved clinical study was conducted by Rosenberg. In this initial study, the gene for neomycin phosphotransferase was introduced into the tumour-infiltrating lymphocytes (TIL) of patients with advanced cancer. Subsequently, he performed clinical trials in which the gene for tumour necrosis factor (TNF) was inserted by retroviral vector into TIL in an effort to increase their therapeutic effectiveness [11]. During the following years, the gene therapy became a very promising approach for the treatment of genetic and oncologic diseases. But serious complication halted the progress of this therapeutic approach. In 1999, Jesse Gelsinger, suffering from a partial deficiency of ornithine transcarbamylase, took part in a gene therapy clinical trial at the University of Pennsylvania. He died due to excessive immune response to a high dose of adenoviral vector [12]. There was a study conducted in Paris, in which 20 children suffering from severe combined immunodeficiency (SCID) took part. They were treated by *ex vivo*–transduced autologous CD34+ haematopoietic progenitor cells. Five of these patients developed T-cell leukaemia [13], and the disease was fatal for one out of these patients [14].

Since then, many steps were taken to improve the safety and efficacy of the gene therapy, and until now, many clinical studies have been conducted.

#### **3. Comparison between retroviral and other viral and non-viral systems**

There are two systems for the delivery of transgene into the cell – viral and non-viral. The nonviral approaches are represented by polymer nanoparticles, lipids, calcium phosphate, electroporation/nucleofection or biolistic delivery of DNA-coated microparticles. The safety is mentioned as the major advantage of non-viral approaches. In general, non-viral delivery of transgene is less effective in comparison to viral systems.

There are several categories of viral vectors. We distinguish two main types of vectors depending on whether the DNA is integrated into chromatin of the host cell or not. Retroviral vectors derived from gammaretroviruses or lentiviruses persist in the nucleus as integrated provirus and reproduce with cell division. Other types of vectors (e.g. those derived from herpesviruses or adenoviruses) remain in cell in the episomal form.

The overview of viral vectors is depicted in Figure 1. Adenoviral and retro/lentiviral vectors are most frequently used in research and gene therapy clinical trials.

As stated above, adenoviral vectors are very popular. They have been used for several decades. Since adenoviruses are non-enveloped dsDNA viruses, they are relatively resistant to chemical and physical agents, which enable them to persist out of host cells and make the work in laboratory easier in comparison to enveloped RNA viruses. They are often used in cancer gene therapy as replication-defective or replication-competent vectors. They infect proliferating as

**Figure 1.** Overview of viral vectors

well as non-dividing cells. In general, adenoviral vectors are considered safe. Since they do not integrate into host DNA, the transduction is transient. The drawback is their immunoge‐ nicity [15-17].

Adeno-associated vectors share with retroviral vectors the ability to integrate into host DNA. Wild type adeno-associated virus integrates into specific site of the chromosome 19 (19q13.3 qter). Recombinant vectors lack this characteristic and the risk of insertional mutagenesis exists. These vectors transduce dividing and non-dividing cells, and the transgene expression is long term. Transduced cells are minimally immunogenic [18].

Herpetic viruses are relatively complex enveloped dsDNA viruses. The vectors have been prepared from Herpes simplex type 1 virus, Epstein–Barr virus or cytomegalovirus. They are less immunogenic in comparison to adenoviruses. The transduction is transient; the drawback of HSV 1-derived vector is the short-term expression of the transgene. Herpes virus-derived vectors are preferentially used in vaccination [19, 20].

Poxviruses are the most complex viruses. Their major advantage lies in the huge cloning capacity. Up to 25 kbp of foreign DNA can be cloned into vaccinia-derived vectors. Similarly to herpes virus-derived vectors, they are popular in the preparation of vaccines including cancer immunotherapy [21, 22].

Baculoviruses, the viruses specific for invertebrates, are not competitors of retroviruses in gene therapy. They have been used for more than 30 years for transduction of insect cells for expression of recombinant proteins. Pseudotyping enables the transduction of mammalian cells [23].

Vectors derived from alfaviruses (ssRNA viruses) are also used in cancer gene therapy and immunotherapy [24, 25].

#### **4. Retroviral vectors**

Retroviruses are relatively complex enveloped RNA viruses with diploid ssRNA genome. Typical feature of retroviruses and retroviral vectors is their ability to integrate into host DNA. Viral RNA is reversibly transcribed and integrated in the form of provirus. They very effec‐ tively cooperate with enzymes of the host cell, and they use it for their own replication and long-term expression of viral proteins. The entry of virus into the host cell is receptordependent [26].

Many types of retroviruses (bovine leukaemia virus, Rous sarcoma virus, lentiviruses and spumaviruses) were used for preparation of vectors. The most popular vectors are constructs based on MoMLV and HIV.

#### **4.1. Gammaretroviral vectors**

well as non-dividing cells. In general, adenoviral vectors are considered safe. Since they do not integrate into host DNA, the transduction is transient. The drawback is their immunoge‐

Adeno-associated vectors share with retroviral vectors the ability to integrate into host DNA. Wild type adeno-associated virus integrates into specific site of the chromosome 19 (19q13.3 qter). Recombinant vectors lack this characteristic and the risk of insertional mutagenesis exists. These vectors transduce dividing and non-dividing cells, and the transgene expression

Herpetic viruses are relatively complex enveloped dsDNA viruses. The vectors have been prepared from Herpes simplex type 1 virus, Epstein–Barr virus or cytomegalovirus. They are less immunogenic in comparison to adenoviruses. The transduction is transient; the drawback of HSV 1-derived vector is the short-term expression of the transgene. Herpes virus-derived

Poxviruses are the most complex viruses. Their major advantage lies in the huge cloning capacity. Up to 25 kbp of foreign DNA can be cloned into vaccinia-derived vectors. Similarly to herpes virus-derived vectors, they are popular in the preparation of vaccines including

Baculoviruses, the viruses specific for invertebrates, are not competitors of retroviruses in gene therapy. They have been used for more than 30 years for transduction of insect cells for expression of recombinant proteins. Pseudotyping enables the transduction of mammalian

Vectors derived from alfaviruses (ssRNA viruses) are also used in cancer gene therapy and

is long term. Transduced cells are minimally immunogenic [18].

vectors are preferentially used in vaccination [19, 20].

cancer immunotherapy [21, 22].

immunotherapy [24, 25].

cells [23].

nicity [15-17].

**Figure 1.** Overview of viral vectors

146 Advances in Molecular Retrovirology

The first MoMLV-based vectors were prepared more than 30 years ago [8, 9], and they are still very popular. The construct is relatively small, and it is possible to achieve high titres in inoculum. The diagram of MoMLV provirus and MoMLV-derived vector is depicted in Figure 2.

**Figure 2. Genome structure of integrated MoMLV and MoMLV-derived vector.** (a) Diagram of MoMLV provirus; (b) Diagram of integrated MoMLV-derived retroviral vector; LTR = long terminal repeats; U3 = unique sequence derived from 3ʹ end of the viral RNA; R = repeated sequence; U5 = unique sequence derived from 5ʹ end of the viral RNA; PBS = primer binding site; SD = splice donor; Ψ = packaging signal; gag = genes for structural proteins; pol = region coding for genes needed for replication of retrovirus; env = genes for envelope proteins; PPT = polypurine tract (according to [27, 28], adjusted).

Contrary to lentiviruses, the packaging system of gammaretroviruses does not require incorporation of sequences overlapping coding region gag, pol or additional genes.

Since MoMLV lacks elements necessary for active transport of genetic information through nuclear membrane, integration of viral DNA is possible only during the mitosis. MoMLVderived vectors transduce only dividing cells. Integration of viral genome is mediated by the pre-integration complex (PIC) consisting of integrase, capsid, p12, proviral DNA and host cell proteins. Proviral DNA is surrounded by two long terminal repeats (LTR), which are com‐ posed of U3, R and U5 regions. Transcription of proviral DNA starts from enhancer/promotor in 5' U3 region [27].

MoMLV constructs can be prepared as replication-competent or replication-deficient.

Gag and env genes are removed in replication-deficient vectors. Gene of interest is cloned in the free space. A typical replication-deficient vector contains packaging signal (ψ), primer binding site (PBS) and LTRs. Viral genes gag, pol and env are cloned on separate expression helper plasmids, and helper cell lines are co-transfected with more plasmids. Stably transfected helper cell lines expressing gag pol (GP) and env were prepared.

Packaging cell lines GP+E-86 and GP-envAM-12 were derived from NIH-3T3 cell line by electroporation of two plasmids. One plasmid contained the gag and pol regions of MoMLV, and the other contained the env region. GP+E-86 cells are used for the production of ecotropic viral particles, and line GP+envAM-12 is used for amphotropic viral particles (explained later) [29]. Expression cassette with gene of interest should not contain introns, internal polyadeny‐ lation signals and large secondary structures, which could interfere with reverse transcriptase. The cloning capacity of retroviral vectors is up to 10 kbps, but the size of transgene significantly influences its expression and viral titre [27].

Envelope protein coded by gene env is responsible for the tropism of MoMLV. Three natural variants of MoMLV were described. They differ in their envelope proteins. Ecotropic MoMLV infects only mouse and rats cells; amphotropic is also able to infect the cells derived from other mammals including human cells. Xenotropic MoMLV was supposed to infect many types of cells excluding murine, but recent studies indicate that it also infects mice and it has the widest tropism among MoMLV [27, 30].

Tropism of the viral vector can be modulated by pseudotyping using a particular envelope protein. The glycoprotein of vesicular stomatitis virus (VSVg) is very popular. It enables broad host range (mammalian and non-mammalian cells). It also has stabilisation properties, and viral particles can be purified by ultracentrifugation. On the other hand, VSVg is recognised by complement, and this fact can decrease the transduction efficacy *in vivo* [31].

#### **4.2. Lentiviral vectors**

In comparison to MoMLV-derived vectors, lentiviral vectors are more complex. Three generations of lentiviral vectors were prepared in order to increase the safety and effica‐ cy of the gene transfer. The viral genome was split into packaging and transfer vectors. The first and second generation are composed of three plasmids; the third generation consists of four plasmids [31]. The main difference between lentiviral vectors and vectors derived from other retroviruses is their ability to infect/transduce quiescent non-dividing cells [32]. They are able to pass through nucleopores into the intact nucleus. The mecha‐ nism of this phenomenon has not been completely clarified; yet, it is known that both viral and cellular proteins participate in this process. In addition to HIV vectors, vectors based on feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV) or equine infectious anaemia virus (EIAV) have also been prepared. Pseudotyping (VSVg is the most common) is typical for lentiviral vectors [31].

#### **4.3. Self-inactivating vectors**

posed of U3, R and U5 regions. Transcription of proviral DNA starts from enhancer/promotor

Gag and env genes are removed in replication-deficient vectors. Gene of interest is cloned in the free space. A typical replication-deficient vector contains packaging signal (ψ), primer binding site (PBS) and LTRs. Viral genes gag, pol and env are cloned on separate expression helper plasmids, and helper cell lines are co-transfected with more plasmids. Stably transfected

Packaging cell lines GP+E-86 and GP-envAM-12 were derived from NIH-3T3 cell line by electroporation of two plasmids. One plasmid contained the gag and pol regions of MoMLV, and the other contained the env region. GP+E-86 cells are used for the production of ecotropic viral particles, and line GP+envAM-12 is used for amphotropic viral particles (explained later) [29]. Expression cassette with gene of interest should not contain introns, internal polyadeny‐ lation signals and large secondary structures, which could interfere with reverse transcriptase. The cloning capacity of retroviral vectors is up to 10 kbps, but the size of transgene significantly

Envelope protein coded by gene env is responsible for the tropism of MoMLV. Three natural variants of MoMLV were described. They differ in their envelope proteins. Ecotropic MoMLV infects only mouse and rats cells; amphotropic is also able to infect the cells derived from other mammals including human cells. Xenotropic MoMLV was supposed to infect many types of cells excluding murine, but recent studies indicate that it also infects mice and it has the widest

Tropism of the viral vector can be modulated by pseudotyping using a particular envelope protein. The glycoprotein of vesicular stomatitis virus (VSVg) is very popular. It enables broad host range (mammalian and non-mammalian cells). It also has stabilisation properties, and viral particles can be purified by ultracentrifugation. On the other hand, VSVg is recognised

In comparison to MoMLV-derived vectors, lentiviral vectors are more complex. Three generations of lentiviral vectors were prepared in order to increase the safety and effica‐ cy of the gene transfer. The viral genome was split into packaging and transfer vectors. The first and second generation are composed of three plasmids; the third generation consists of four plasmids [31]. The main difference between lentiviral vectors and vectors derived from other retroviruses is their ability to infect/transduce quiescent non-dividing cells [32]. They are able to pass through nucleopores into the intact nucleus. The mecha‐ nism of this phenomenon has not been completely clarified; yet, it is known that both viral and cellular proteins participate in this process. In addition to HIV vectors, vectors based on feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV) or equine infectious anaemia virus (EIAV) have also been prepared. Pseudotyping (VSVg is the most

by complement, and this fact can decrease the transduction efficacy *in vivo* [31].

MoMLV constructs can be prepared as replication-competent or replication-deficient.

helper cell lines expressing gag pol (GP) and env were prepared.

influences its expression and viral titre [27].

common) is typical for lentiviral vectors [31].

tropism among MoMLV [27, 30].

**4.2. Lentiviral vectors**

in 5' U3 region [27].

148 Advances in Molecular Retrovirology

The risk of insertional mutagenesis is a drawback of retroviral vectors. With the purpose of increasing the safety of gene therapy, self-inactivating (SIN) vectors were prepared in the 1980s. SIN vectors have a deletion in the 3' U3 region, where promotor and enhancer sequences occur. This deletion is copied into 5' LTR during the reverse transcription, and virus becomes free of LTR-bound promotor activity. Transcription control is under the chosen cloned promotor [33].

SIN vectors are characterised by decreased risk of insertional mutagenesis; the vector is not activated via infection by another retrovirus, and the internal promotor is autonomous. Controlled/inducible or cell-specific expression of transgene can be achieved based on the chosen promotor [33]. The use of tetracycline (Tet)-inducible system was published in the 1990s [34], and until now it is being used for enhanced expression of an exogenous gene in a celltype-specific manner.

#### **4.4. Replication-competent retroviral vectors**

Retroviral vectors can be constructed as replication-defective to transduce target cells and enable long-term expression of transgene (immunology disorders, genetic diseases), or they carry transgenes inducing cell death (cancer gene therapy). On the other hand, the replicationcompetent vectors (RCV) are prepared in order to replicate in the target (tumour) cells. Their progeny infects surrounding malignant cells. Since the targeting is an inevitable characteristic of RCV, they can be engineered to express ligands to tumour cell-specific markers. The advantage of MoMLV-derived vectors is their natural preference to tumour cells [35]. MoMLV is unable to infect quiescent cells, making them suitable vehicles for the treatment of brain tumours. RCV with suicide gene mediate synchronised cell killing after prodrug administra‐ tion, and due to their stable integration into DNA of infected cells, residual cancer cells serve as a reservoir for long-term viral persistence even when they migrate to new sites. Multiple cycles of prodrug administration to achieve improved treatment efficacy are possible [36].

Although gammaretroviral and lentiviral vectors are derived from the same viral family, they differ in some characteristics. Advantages of gammaretroviral vectors reside in the complete absence of viral gene remnants in the transfer vector, efficient pseudotyping and the lack of mobilisation by human-infectious viruses. In comparison to HIV-derived vectors, there are also minor concerns related to potential seroconversion *in vivo*. Lentiviral vectors are clearly superior for the ability to transduce non-dividing cells. Both types of vectors are equally potent in terms of expression properties when containing similar internal expression cassettes [37]. Compared to vectors derived from non-integrating viruses, retroviral vectors possess the risk of insertional mutagenesis. Gammaretroviral vectors preferentially integrate close to tran‐ scription start sites and CpG islands, which are enriched in gene-regulatory elements. Lentiviral vectors prefer integration inside of the transcription units of actively transcribed genes [38].

Despite the promise for success in the clinic, one major drawback of the retrovirus-based vector is that any unintended insertion events from the therapy can potentially lead to deleterious effects or it can cause an abnormal expression of nearby host genes driven by the enhancer of the inserted viral DNA in patients, as demonstrated by the development of malignancies in both animal and human studies. The better prediction of the integration sites by elucidation of this mechanism might lead to the development of retroviral vectors capable of selective integration. This understanding could provide the ultimate solution to the problems of insertional mutagenesis [39]. The definition of the precise mechanism of the retroviral preintegration complex is required. Many efforts have been made in designing modified integra‐ ses with sequence-specific integration capability, which can be accomplished by rational modification of the protein or by using the directed evolution approach [40]. One approach to directing integration into predetermined DNA sites is fusing integrase to a sequence-specific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner [41]. Efforts to engineer integrase to recognise specific target DNA sequences within the host genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting. Insertion of a lentiviral vector via virionassociated Cre protein, capable of directing site-specific insertion of a gene in the vector, into a defined loxP site in the host genome was described [42].

A detailed study of the vector integration sites performed on haematopoietic stem cells by Aiuti *et al.* [43] concluded that lentiviral gene therapy was safer than retroviral gene therapy, and lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months follow-up. Also, the so-called integration-deficient lentiviral vectors (IDLVs) can be produced through the use of integrase mutations that specifically prevent proviral integration. These lentiviral episomes lack replication signals and are gradually lost by dilution in dividing cells, but are stable in quiescent cells. Compared to integrating lentivectors, IDLVs have a greatly reduced risk of causing insertional mutagenesis [44].

#### **4.5. Overview of preparation of the gammaretroviral vector**

Recently, many viral cloning systems are available. Transgene is cloned either directly or via a bacterial intermediate. Circular dsDNA is found at the beginning of the process. It contains viral sequences including LTR necessary for the integration, genes coding for antibiotic resistance and target sequences for different restriction endonucleases (multicloning sites). The first part of vector construction takes place in bacteria; therefore, the bacterial origin of replication (ORI) is a necessary component. The gene of interest is cloned into the vector, then propagated in bacteria and verified by sequencing. Subsequently, the packaging cell line is transfected, and the sequences between LTRs are integrated into host-cell DNA. Vectorcontaining cells are selected via antibiotic resistance. In order to increase the titre of the viral vector, ecotropic and amphotropic packaging cell lines can be used. First, the ecotropic cell line is transfected, and then the amphotropic packaging cells are transduced by viruscontaining cultivation supernatant from ecotropic cells. The 'ping-pong' method – mutual exchange of virus-containing medium between ecotropic and amphotropic cells – is performed to further increase the viral titre. The viral titre is determined and cultivation supernatant from transduced cells is collected for transduction of target cells. Transgene-containing cells are selected via antibiotic resistance (Figure 3).

**Figure 3. The preparation of replication-defective retrovirus vector.** (a) Circular dsDNA construct, which is transfect‐ ed to bacteria; (b) particular steps of vector preparation: 1. cloning of desired gene into the vector; 2. transformation of bacteria; 3. selection of bacterial clones with desired transgene (NeoR); 4. verification of cloned gene by sequencing; 5. multiplication of desirable clone; 6. purification of plasmid DNA; 7. transfection via packaging cell line; 8. selection of transfected cells (NeoR); 9. virus titration; 10. ping-pong; 11. virus titration after ping-pong; 12. transduction of target cells; 13. selection of transduced target cells [NeoR/G418R – resistance of bacteria to neomycin and mammalian cells' resistance to geneticin (G418) coding by *NeoR* gene].

#### **5. Gene therapy**

effects or it can cause an abnormal expression of nearby host genes driven by the enhancer of the inserted viral DNA in patients, as demonstrated by the development of malignancies in both animal and human studies. The better prediction of the integration sites by elucidation of this mechanism might lead to the development of retroviral vectors capable of selective integration. This understanding could provide the ultimate solution to the problems of insertional mutagenesis [39]. The definition of the precise mechanism of the retroviral preintegration complex is required. Many efforts have been made in designing modified integra‐ ses with sequence-specific integration capability, which can be accomplished by rational modification of the protein or by using the directed evolution approach [40]. One approach to directing integration into predetermined DNA sites is fusing integrase to a sequence-specific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner [41]. Efforts to engineer integrase to recognise specific target DNA sequences within the host genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting. Insertion of a lentiviral vector via virionassociated Cre protein, capable of directing site-specific insertion of a gene in the vector, into

A detailed study of the vector integration sites performed on haematopoietic stem cells by Aiuti *et al.* [43] concluded that lentiviral gene therapy was safer than retroviral gene therapy, and lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months follow-up. Also, the so-called integration-deficient lentiviral vectors (IDLVs) can be produced through the use of integrase mutations that specifically prevent proviral integration. These lentiviral episomes lack replication signals and are gradually lost by dilution in dividing cells, but are stable in quiescent cells. Compared to integrating lentivectors, IDLVs have a greatly reduced risk of

Recently, many viral cloning systems are available. Transgene is cloned either directly or via a bacterial intermediate. Circular dsDNA is found at the beginning of the process. It contains viral sequences including LTR necessary for the integration, genes coding for antibiotic resistance and target sequences for different restriction endonucleases (multicloning sites). The first part of vector construction takes place in bacteria; therefore, the bacterial origin of replication (ORI) is a necessary component. The gene of interest is cloned into the vector, then propagated in bacteria and verified by sequencing. Subsequently, the packaging cell line is transfected, and the sequences between LTRs are integrated into host-cell DNA. Vectorcontaining cells are selected via antibiotic resistance. In order to increase the titre of the viral vector, ecotropic and amphotropic packaging cell lines can be used. First, the ecotropic cell line is transfected, and then the amphotropic packaging cells are transduced by viruscontaining cultivation supernatant from ecotropic cells. The 'ping-pong' method – mutual exchange of virus-containing medium between ecotropic and amphotropic cells – is performed to further increase the viral titre. The viral titre is determined and cultivation supernatant from transduced cells is collected for transduction of target cells. Transgene-containing cells are

a defined loxP site in the host genome was described [42].

**4.5. Overview of preparation of the gammaretroviral vector**

causing insertional mutagenesis [44].

150 Advances in Molecular Retrovirology

selected via antibiotic resistance (Figure 3).

Treatment of genetic diseases and cancer gene therapy are the main targets of recent gene therapy. They belong to serious diseases, which are difficult to treat or are incurable using conventional treatment, or the treatment is accompanied by severe adverse effects.

Over 60% of ongoing gene therapy clinical trials represent cancer treatment followed by monogenetic and cardiovascular diseases [11]. Two approaches of gene therapy are defined: (i) The *ex vivo* method is characterised by the collection of target cells from the organism, genetic modification and subsequent administration to the patient. (ii) The *in vivo* method is charac‐ terised by the direct administration of therapeutic gene to the patient.

#### **5.1. Treatment of genetic, immunologic and other non-oncologic diseases**

Genetic as well as age-related diseases can be treated by gene therapy. They are caused by deficiency or aberrant expression of one or more gene(s). Patients suffering from severe combined immunodeficiency (SCID) – devastating disorder of adapted immunity – are not able to defend against infections. The term SCID covers several genetic defects. Adenosine deaminase (ADA) deficiency was the first SCID condition for which a genetic and molecular cause was identified [45]. The patients suffering from ADA-SCID are ideal candidates for gene therapy, when haematopoietic cells are transduced by a gene encoding adenosine deaminase. A retroviral vector carrying ADA was the first construct to contain a therapeutic gene (Rosen‐ berg transduced cells with neomycin resistance gene to track them *in vivo*) used in an FDAapproved clinical trial. Two children were treated [46]. SCID-X1 is characterised by various mutations in the gene encoding interleukin 2 receptor-γ (IL2RG) [47, 48]. Transduction of functional IL2RG restores expression of functional interleukin 2 receptor-γ. It was demon‐ strated that gene therapy for primary immunodeficiencies is an effective treatment modality providing long-term clinical benefit for patients. Lentiviral vectors contributed significantly to this achievement [49]. Haematopoietic stem cells were engineered *ex vivo* and administered to the patient. Long-term T-cell reconstitution was achieved in patients suffering from ADA-SCID and SCID-X [50].

Age-related macular degeneration is accompanied with excessive vascularisation in which vascular endothelial growth factor (VEGF) takes place. Its function can be inhibited by retrovirally delivered antiangiogenic factors such as angiostatin, endostatin or extracellular domain of VEGF receptor. Vectors are administered into vitreous body or under retina. Antiangiogenic genes are also used in cancer gene therapy [51-53].

Epidermolysis bullosa is a group of devastating skin disorders. Mutations in the COL7A1 gene result in the absence or dysfunction of type VII collagen protein and cause recessive dystrophic epidermolysis bullosa (RDEB). Collagen VII expression has been restored by retroviral and lentiviral vectors carrying COL7A1 gene. Long-term expression of transgene in keratinocytes, fibroblasts or epidermal stem cells was achieved [54]. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) are the cause of junctional epidermolysis bullosa. Retrovirally transduced epidermal stem cells have been used for preparation of epidermal grafts. Analysis revealed that synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epider‐ mis that remained stable at least for 1 year [55].

The treatment of congenital disorders of liver metabolism is currently limited, and prognosis of patients suffering from Crigler–Najjar syndrome, urea cycle disorders, familial hypercho‐ lesterolemia and primary hyperoxaluria type 1 is unfavourable. In comparison to the liver transplantation, *ex vivo* gene therapy offers a less invasive method without the need for lifelong immunosuppression [56].

Homozygous mutation in LDL receptor causes familial hypercholesterolemia. The gene therapy approach for treatment of this lethal disease was developed. Grossman *et al*. [57] described the treatment of a 29-year-old woman by *ex vivo* gene therapy. Autologous hepato‐ cytes isolated from the patient were genetically corrected with recombinant retroviruses carrying the LDL receptor and subsequently reimplanted. The patient's LDL/HDL ratio improvements have remained stable for the duration of the treatment (18 months). *Ex vivo* gene therapy using modified haematopoietic stem cells has generated encouraging results for treatment of multisystemic lysosomal storage disorders [58]. Retroviral and lentiviral vectortransduced bone marrow-derived cells overexpressing lysosomal enzymes can migrate into the central nervous system (CNS) and mediate cross-correction of the neighbouring brain cells. This approach has resulted in excellent outcomes, preventing the development of clinical manifestations in metachromatic leukodystrophy. According to a study by Cartier *et al*. [59], patients with peroxisomal disorder X-linked adrenoleukodystrophy treated with *ex vivo* lentiviral vector-mediated gene therapy have also exhibited clinical benefits.

Based on the significant progress made to date, in spite of the expected setbacks of all drug development efforts, gene therapy for liver metabolic disorders is becoming a viable option for treatment in future clinical trials.

#### **5.2. Cancer gene therapy**

berg transduced cells with neomycin resistance gene to track them *in vivo*) used in an FDAapproved clinical trial. Two children were treated [46]. SCID-X1 is characterised by various mutations in the gene encoding interleukin 2 receptor-γ (IL2RG) [47, 48]. Transduction of functional IL2RG restores expression of functional interleukin 2 receptor-γ. It was demon‐ strated that gene therapy for primary immunodeficiencies is an effective treatment modality providing long-term clinical benefit for patients. Lentiviral vectors contributed significantly to this achievement [49]. Haematopoietic stem cells were engineered *ex vivo* and administered to the patient. Long-term T-cell reconstitution was achieved in patients suffering from ADA-

Age-related macular degeneration is accompanied with excessive vascularisation in which vascular endothelial growth factor (VEGF) takes place. Its function can be inhibited by retrovirally delivered antiangiogenic factors such as angiostatin, endostatin or extracellular domain of VEGF receptor. Vectors are administered into vitreous body or under retina.

Epidermolysis bullosa is a group of devastating skin disorders. Mutations in the COL7A1 gene result in the absence or dysfunction of type VII collagen protein and cause recessive dystrophic epidermolysis bullosa (RDEB). Collagen VII expression has been restored by retroviral and lentiviral vectors carrying COL7A1 gene. Long-term expression of transgene in keratinocytes, fibroblasts or epidermal stem cells was achieved [54]. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) are the cause of junctional epidermolysis bullosa. Retrovirally transduced epidermal stem cells have been used for preparation of epidermal grafts. Analysis revealed that synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epider‐

The treatment of congenital disorders of liver metabolism is currently limited, and prognosis of patients suffering from Crigler–Najjar syndrome, urea cycle disorders, familial hypercho‐ lesterolemia and primary hyperoxaluria type 1 is unfavourable. In comparison to the liver transplantation, *ex vivo* gene therapy offers a less invasive method without the need for lifelong

Homozygous mutation in LDL receptor causes familial hypercholesterolemia. The gene therapy approach for treatment of this lethal disease was developed. Grossman *et al*. [57] described the treatment of a 29-year-old woman by *ex vivo* gene therapy. Autologous hepato‐ cytes isolated from the patient were genetically corrected with recombinant retroviruses carrying the LDL receptor and subsequently reimplanted. The patient's LDL/HDL ratio improvements have remained stable for the duration of the treatment (18 months). *Ex vivo* gene therapy using modified haematopoietic stem cells has generated encouraging results for treatment of multisystemic lysosomal storage disorders [58]. Retroviral and lentiviral vectortransduced bone marrow-derived cells overexpressing lysosomal enzymes can migrate into the central nervous system (CNS) and mediate cross-correction of the neighbouring brain cells. This approach has resulted in excellent outcomes, preventing the development of clinical manifestations in metachromatic leukodystrophy. According to a study by Cartier *et al*. [59],

Antiangiogenic genes are also used in cancer gene therapy [51-53].

mis that remained stable at least for 1 year [55].

immunosuppression [56].

SCID and SCID-X [50].

152 Advances in Molecular Retrovirology

Cancer is a complex disease accompanied by progressive accumulation of genetic and epigenetic alterations, which enables the cell to escape from cell and environmental control. Conventional treatment consists of surgery, chemotherapy and radiotherapy. Despite progress in the treatment, it is ineffective or accompanied by severe adverse effects in many cases; therefore, novel approaches are needed. Recently, research has been focusing on targeted therapy. Gene therapy – classical or mediated by cellular vehicles – is the promising approach, primarily for patients suffering from glioblastoma multiforme (GBM), neuroblastoma, metastatic melanoma and other metastatic cancers. As stated above, cancer treatment is recently the most elaborated area of gene therapy.

The treatment is focused on suppression of activated oncogenes, restoration of expression of tumour-suppressor genes, activation of anti-tumour immunity and inhibition of angiogenesisor metastatic potential-suppressing genes. The separate group is represented by genes inducing autodestruction of tumour – 'toxic' genes or genes coding for enzymes converting non-toxic prodrug to toxic product [60].

Prodrug-converting genes are also known as 'suicide genes'. They are of viral, bacterial or yeast origin, and they do not have the equivalent in mammalian cells. Mammalian cells obtain the ability to utilise a new substrate after transduction – an inactive compound is converted to chemotherapeutics. The apoptosis is induced in transduced cells; therefore, this approach is called suicide gene therapy. Bystander effect is the phenomenon of suicide gene therapy. Toxic metabolites released from transduced cells are received by the surrounding bystander cells and mediate toxic effect in them [61]. This targeted chemotherapy is considerate to the organism because chemotherapeutic agent is present only in the tumour vicinity – the site where (systemically administered) prodrug and therapeutic enzyme meet together.

Herpes simplex virus thymidine kinase (HSVtk) is one of the most frequently used therapeutic genes. Its affinity to nucleotide analogue ganciclovir (GCV) is approximately 1000 times higher than mammalian thymidine kinase. HSVtk phosphorylates GCV to GCV-monophosphate (GCV-P), which is subsequently phosphorylated by cellular kinases and incorporated into replicating DNA instead of guanosine triphosphate. GCV lacks deoxyribose at 3'OH and bound between carbons 2' and 3', which are necessary for the elongation of DNA chain. Incorporation of GCV-3P yields into termination of DNA synthesis and subsequent cell death – preferentially apoptosis, advisable cell death in cancer treatment. GCV becomes a charged molecule after phosphorylation, and it is not able to diffuse across the membrane. As stated above, the bystander effect is an important phenomenon in contributing to the efficacy of gene therapy. Gap junctional intercellular communication (GJIC) is necessary for transport of phosphorylated GCV. GJIC is often a limiting factor of HSVtk/GCV system because many cells are defective in expression of the connexins, which are the major components of gap junctions. Transduction of connexin gene can improve the efficacy of the treatment [62].

The second most frequently used system in suicide gene therapy is cytosine deaminase (CD) derived from bacteria (bCD) or yeasts (yCD) combined with prodrug 5-fluorycytosine (5-FC), which is used in conventional antimycotic therapy. Yeast CD is 15 times more efficient in comparison to its bacterial counterpart [63].

Transduced cell is able to convert non-toxic 5-FC to conventionally used chemotherapeutic 5 fluorouracil (5-FU). More active molecules arise by the metabolism of 5-FU, and the synthesis of both DNA and RNA is impaired. Thymidylate synthase (TS) is the key target enzyme in 5- FC/5-FU treatment. 5-fluorouridine monophosphate, one of the active metabolites of 5-FU, binds irreversibly to TS, and starvation for thymine leads to inhibition of DNA synthesis. 5 fluorothymidine triphosphate, another metabolite, impairs RNA by incorporation instead of UTP [64].

The efficacy of the CD/5-FC approach can be increased by the addition of another enzyme, bacterial or yeast-derived uracil phosphoribosyl transferase (UPRT), which supplements low expression of mammalian orotate phosphoribosyl transferase important for the activation of 5-FU. It also continuously utilises 5-FU supporting its synthesis [65]. CD and UPRT can be cloned separately or as a synthetic fusion gene CD::UPRT.

Purine nucleoside phosphatase (PNP) is an *E. coli*-derived enzyme, which activates fludara‐ bine. PNP-transduced cells convert fludarabine into metabolites, which are highly toxic for proliferating as well as for quiescent cells, because they inhibit ATP-dependent reactions. The metabolism of nucleic acid and proteins is impaired.

When the therapeutic gene is inserted into a replication-defective vector, it is expressed only in the transduced cell, and surrounding cells are affected only via the bystander effect. On the other hand, the replication-competent vectors replicate in target cells, and despite the by‐ stander killing, they are able to infect their neighbours and spread the transgene. RCV have been constructed for treatment of aggressive tumours.

Promising results by MoMLV-derived replication-competent vector carrying cytosine deam‐ inase were achieved on orthotopic glioblastoma model. Considerable infection of target cells, virus spread and significant bystander effect were demonstrated [66]. Based on promising preclinical results, a phase I/II clinical study is ongoing using the replication-competent cytosine deaminase-expressing retroviral vector to patients with recurrent or progressive grade III or grade IV gliomas (NCT01985256) [67].

It is important to note that despite of very promising preclinical data, many clinical studies failed because of low efficiency of the transfer of genetic information into target cells, insuffi‐ cient infiltration of target tissue by the vector or low expression of transgene in tumour. The identification of mesenchymal stromal cells (MSC) and the discovery of their high affinity to tumour tissue facilitate important improvement in the cancer gene therapy.

MSC were originally isolated from bone marrow and characterised as rare non-haematopoietic population with clonogenic capacity and plastic adherence [68]. They have the self-renewal potential and are able to differentiate into specialised progeny [69-72]. Bone marrow is the most popular source of MSC, but adipose tissue is also very suitable because of its accessibility. Moreover, the frequency of MSC in the adipose tissue is 500 times higher in comparison to bone marrow. MSC can also be isolated from umbilical cord, dental pulp and different connective tissues. They serve as a source of regenerative cells in fractures, inflammation and necrosis. The injured tissue produces chemotactic signals which attract MSC [73]. Tumour can be compared to a wound that never heals [74]. Many tumours produce chemotactic signals which attract MSC [75-77]. It was demonstrated that MSC-derived cells are a component of tumour stroma, and they can support proliferation and vascularisation of malignant tissue [78]. The natural affinity of MSC to malignant tissue can be used for targeted therapy. They can be used as delivery vehicles in cancer gene therapy [28, 79]. The approach can be compared to the Trojan horse. MSC are able to pass across the endothelium, enter the blood stream and engraft in the tumour. Therefore, genetically engineered MSC can be administered intrave‐ nously, and they reach the target site. This enables to treat the disseminated tumours and metastases. MSC even cross the blood-brain barrier [80, 81]. In this regard, the MSC-mediated cancer gene therapy is superior to the 'classic' cancer gene therapy. On the other hand, it is also necessary to note that the therapy by genetically engineered MSC is limited by the fact that tumours differ in the attractiveness for MSC. Many paracrine factors are involved in MSC – tumour cell signalling. The SDF-1α and CXCR4 (CXCL12) signalling seem to play an important role in homing of stromal cells [76, 77].

phosphorylated GCV. GJIC is often a limiting factor of HSVtk/GCV system because many cells are defective in expression of the connexins, which are the major components of gap junctions.

The second most frequently used system in suicide gene therapy is cytosine deaminase (CD) derived from bacteria (bCD) or yeasts (yCD) combined with prodrug 5-fluorycytosine (5-FC), which is used in conventional antimycotic therapy. Yeast CD is 15 times more efficient in

Transduced cell is able to convert non-toxic 5-FC to conventionally used chemotherapeutic 5 fluorouracil (5-FU). More active molecules arise by the metabolism of 5-FU, and the synthesis of both DNA and RNA is impaired. Thymidylate synthase (TS) is the key target enzyme in 5- FC/5-FU treatment. 5-fluorouridine monophosphate, one of the active metabolites of 5-FU, binds irreversibly to TS, and starvation for thymine leads to inhibition of DNA synthesis. 5 fluorothymidine triphosphate, another metabolite, impairs RNA by incorporation instead of

The efficacy of the CD/5-FC approach can be increased by the addition of another enzyme, bacterial or yeast-derived uracil phosphoribosyl transferase (UPRT), which supplements low expression of mammalian orotate phosphoribosyl transferase important for the activation of 5-FU. It also continuously utilises 5-FU supporting its synthesis [65]. CD and UPRT can be

Purine nucleoside phosphatase (PNP) is an *E. coli*-derived enzyme, which activates fludara‐ bine. PNP-transduced cells convert fludarabine into metabolites, which are highly toxic for proliferating as well as for quiescent cells, because they inhibit ATP-dependent reactions. The

When the therapeutic gene is inserted into a replication-defective vector, it is expressed only in the transduced cell, and surrounding cells are affected only via the bystander effect. On the other hand, the replication-competent vectors replicate in target cells, and despite the by‐ stander killing, they are able to infect their neighbours and spread the transgene. RCV have

Promising results by MoMLV-derived replication-competent vector carrying cytosine deam‐ inase were achieved on orthotopic glioblastoma model. Considerable infection of target cells, virus spread and significant bystander effect were demonstrated [66]. Based on promising preclinical results, a phase I/II clinical study is ongoing using the replication-competent cytosine deaminase-expressing retroviral vector to patients with recurrent or progressive

It is important to note that despite of very promising preclinical data, many clinical studies failed because of low efficiency of the transfer of genetic information into target cells, insuffi‐ cient infiltration of target tissue by the vector or low expression of transgene in tumour. The identification of mesenchymal stromal cells (MSC) and the discovery of their high affinity to

tumour tissue facilitate important improvement in the cancer gene therapy.

Transduction of connexin gene can improve the efficacy of the treatment [62].

comparison to its bacterial counterpart [63].

154 Advances in Molecular Retrovirology

cloned separately or as a synthetic fusion gene CD::UPRT.

metabolism of nucleic acid and proteins is impaired.

been constructed for treatment of aggressive tumours.

grade III or grade IV gliomas (NCT01985256) [67].

UTP [64].

Besides MSC, other cellular vehicles can be transduced and used in cancer gene therapy. Neural progenitors or neural stem cells isolated from brain tissue are used as delivery vehicles in targeted therapy of aggressive tumours of central nervous system [82].

The ideal candidates of cellular therapy for clinical use are the cells harvested without difficulty, which can be processed *ex vivo* very efficiently and afterwards transplanted. The unique biological features of MSC predetermine them as valuable gene carriers for therapeutic approaches. MSC can be easily transduced with retroviral and lentiviral vectors, which is a key prerequisite for the introduction and durable expression of marker and/or therapeutic genes within the tumour environment after homing to target tissues [83, 84].

The immunophenotype andthe ability todifferentiate arenot affectedby transduction.Inorder to address the safety of retrovirally transduced MSC, many studies have been performed. Particular transgene can give a proliferative advantage, but it does not preclude the entering of cells tosenescence andhasnoimpactonthe safetyof cancergene therapymediatedbyMSC[85].

The combination of cellular and gene therapy provides a unique opportunity to bypass the obstacles connected with direct viral delivery of the transgene. Cell vehicle protects the vector from the immune surveillance and supports targeting of a therapeutic molecule to the tu‐ mour [86].

Cell-mediated gene therapy is based on the bystander effect. The suicide effect is not the main goal; neighbouring bystander cells should be impaired at first. Therefore, it is more appropriate to use the term 'prodrug-converting gene' instead of 'the suicide gene'.

The simple retroviral plasmid pJZ308 derived from Moloney murine leukaemia virus [87] was used for delivery of yeast *cytosine deaminase::uracil phosphoribosyltransferase* (CD::UPRT) and *Herpes simplex* virus-thymidine kinase (HSVtk) prodrug-converting genes into adipose tissuederived MSC. The retroviral transduction of AT-MSC by CD gene was published for the first time in 2007. In this pilot study, the capability of AT-MSC expressing fusion yeast CD::UPRT gene in combination with prodrug 5-fluorocytosine (5-FC) to eradicate human colon carcinoma cells HT-29 *in vitro*, and their significant role in inhibition of tumour growth in a therapeutic paradigm *in vivo* were demonstrated [88]. A number of published papers reported the cytotoxic efficiency of CD::UPRT-MSC/5-FC enzyme/prodrug therapeutic system, both *in vitro* and *in vivo*, in the treatment of experimental prostate tumour [89, 90], melanoma [91, 92] and medullary thyroid carcinoma [93, 94]. The 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models are suggested to be used according to the study performed on melanoma model [95]. Contrary to 5-FU, 5-FC is able to cross the blood-brain barrier, thus making this enzyme/prodrug approach suitable for the treatment of CNS tumours [96], which was proved on malignant glioma model [97]. The complete regression of glioblastoma simulating clinical therapeutic scenario was demonstrat‐ ed by Altaner *et al.* [98].

AT-MSC were shown to form gap junctional intercellular communication with glioblastoma cell lines, thus rendering them suitable vehicles for the enzyme/prodrug therapy system HSVtk/GCV relying on transport of polar metabolites [99]. AT-MSC transduced by this suicide gene HSVtk also via lentiviral vector proved strong candidates of gene therapy for U-87 derived model of glioblastoma multiforme [100].

Efficacy of gene-directed enzyme/prodrug therapy can be improved by the combination of individual systems. Matuskova *et al*. [101] demonstrated various levels of synergy depending on tested cell line and experimental set-up. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with both prodrugs, 5-FC and GCV, inhibited growth of experi‐ mental lung metastases derived from human breast adenocarcinoma cells.

MSC were also retrovirally transduced to stably express an exogenous gene encoding the therapeutic agent hTNFα whose effect was tested on tumour cell lines of different origins. Coinjection of such therapeutic cells with melanoma cells inhibited the tumour mass growth up to 97.5% *in vivo* [102].

MSC isolated from the Wharton's jelly of the human umbilical cord were lentivirally trans‐ duced by gene carrying the soluble human tumour necrosis factor-related apoptosis-inducing ligand (sTRAIL). The specific expression of the transgene in the tumour was ensured by alphafetoprotein promoter. Significant therapeutic effect was observed on orthotopic hepatocarci‐ noma model established on athymic mice, and the treatment was even more efficient in combination with 5-fluorouracil [103].

The effectiveness of therapeutic system using TRAIL expression from bone marrow-derived MSC with significantly increased survival of nude mice was noted as suitable for use in the prevention of the recurrence of hepatocellular carcinoma after radiofrequency ablation [104].

A different therapeutic strategy comprises carboxyl esterase (CE), an enzyme hydrolysing prodrug Irinotecan. Hong *et al*. [105] transduced neural stem cells by this gene, which led to the development of a novel strategy for delivering therapeutic genes to brain tumours. The significant inhibition of the growth of human non-small-cell lung adenocarcinoma cells was achieved for these lung cancer brain metastases also *in vivo*.

PNP-transduced AT-MSC were tested for treatment of ovarian cancer in immunodeficient mice model (unpublished data). Cell vehicles were also retrovirally transduced by interleukincoding genes (IL-2, IL-4, IL-12, IL-23) and interferon-β in order to treat primary or metastatic brain tumours [96].

As stated above, MSC are the long-term reservoir for tissue regeneration. They are naturally radio- and chemo-resistant [73, 106, 107]. Despite being equipped by enzymes and efflux mechanisms enabling resistance to chemotherapeutics, their resistance is not absolute. The transgene or metabolite activated by prodrug-converting gene also affects MSC, which after a certain time undergo cell death [108]. In the context of tumour-promoting potential of MSC [76], this fact should be considered as the advantage improving the safety of MSC-mediated gene therapy. It was shown that expression of yCD::UPRT transgene sensitises MSC to 5-FC, and its expression as well as the expression of HSVtk lead to suicide effect of therapeutic cells in the presence of GCV [108]. As shown on neural stem cells, if they are co-expressed together, the effect is even stronger [109].

Promising preclinical data enabled the approval of clinical trials mediated by engineered cellular vehicles. Patients suffering from aggressive, by conventional approaches incurable tumours can be included. The protocol for the first clinical study utilising genetically engi‐ neered MSC was published in 2015. Patients suffering from advanced, recurrent or metastatic gastrointestinal or hepatopancreatobiliary adenocarcinoma will be treated by autologous retrovirally transduced bone marrow-derived MSC. The gammaretroviral self-inactivating vector carrying HSVtk will be used [110].

To conclude, it is important to note that despite many clinical studies, the gene therapy is in the early stage of clinical use. For now, it presents an experimental approach. Besides clinical efficacy, safety is the crucial criterion of gene therapy. It is undisputed that retroviral vectors are indispensable tools for genetic modification, and they have the potential to significantly contribute to the improvement in targeted treatment of immunologic, oncologic and genetic disorders.

#### **Acknowledgements**

The simple retroviral plasmid pJZ308 derived from Moloney murine leukaemia virus [87] was used for delivery of yeast *cytosine deaminase::uracil phosphoribosyltransferase* (CD::UPRT) and *Herpes simplex* virus-thymidine kinase (HSVtk) prodrug-converting genes into adipose tissuederived MSC. The retroviral transduction of AT-MSC by CD gene was published for the first time in 2007. In this pilot study, the capability of AT-MSC expressing fusion yeast CD::UPRT gene in combination with prodrug 5-fluorocytosine (5-FC) to eradicate human colon carcinoma cells HT-29 *in vitro*, and their significant role in inhibition of tumour growth in a therapeutic paradigm *in vivo* were demonstrated [88]. A number of published papers reported the cytotoxic efficiency of CD::UPRT-MSC/5-FC enzyme/prodrug therapeutic system, both *in vitro* and *in vivo*, in the treatment of experimental prostate tumour [89, 90], melanoma [91, 92] and medullary thyroid carcinoma [93, 94]. The 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models are suggested to be used according to the study performed on melanoma model [95]. Contrary to 5-FU, 5-FC is able to cross the blood-brain barrier, thus making this enzyme/prodrug approach suitable for the treatment of CNS tumours [96], which was proved on malignant glioma model [97]. The complete regression of glioblastoma simulating clinical therapeutic scenario was demonstrat‐

AT-MSC were shown to form gap junctional intercellular communication with glioblastoma cell lines, thus rendering them suitable vehicles for the enzyme/prodrug therapy system HSVtk/GCV relying on transport of polar metabolites [99]. AT-MSC transduced by this suicide gene HSVtk also via lentiviral vector proved strong candidates of gene therapy for U-87-

Efficacy of gene-directed enzyme/prodrug therapy can be improved by the combination of individual systems. Matuskova *et al*. [101] demonstrated various levels of synergy depending on tested cell line and experimental set-up. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with both prodrugs, 5-FC and GCV, inhibited growth of experi‐

MSC were also retrovirally transduced to stably express an exogenous gene encoding the therapeutic agent hTNFα whose effect was tested on tumour cell lines of different origins. Coinjection of such therapeutic cells with melanoma cells inhibited the tumour mass growth up

MSC isolated from the Wharton's jelly of the human umbilical cord were lentivirally trans‐ duced by gene carrying the soluble human tumour necrosis factor-related apoptosis-inducing ligand (sTRAIL). The specific expression of the transgene in the tumour was ensured by alphafetoprotein promoter. Significant therapeutic effect was observed on orthotopic hepatocarci‐ noma model established on athymic mice, and the treatment was even more efficient in

The effectiveness of therapeutic system using TRAIL expression from bone marrow-derived MSC with significantly increased survival of nude mice was noted as suitable for use in the prevention of the recurrence of hepatocellular carcinoma after radiofrequency ablation [104].

mental lung metastases derived from human breast adenocarcinoma cells.

ed by Altaner *et al.* [98].

156 Advances in Molecular Retrovirology

to 97.5% *in vivo* [102].

combination with 5-fluorouracil [103].

derived model of glioblastoma multiforme [100].

Studies mentioned in this chapter were also supported by the Slovak Research and Develop‐ ment Agency under contract nos. APVV-0052-12 and APVV-0230-11, by VEGA grant nos. 2/0171/13, 2/0087/15, d2/0130/13 and by the Slovak Cancer Research Foundation.

#### **Author details**

Miroslava Matuskova\* and Erika Durinikova

\*Address all correspondence to: exonmigu@savba.sk

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia

#### **References**


[12] Stolberg SG: The biotech death of Jesse Gelsinger. The New York Times Magazine. 1999:136-40, 49-50.

**Author details**

**References**

Miroslava Matuskova\*

158 Advances in Molecular Retrovirology

1952;64(5):679-99.

1961;13(2):158-63.

1964;23(4):486-94.

10.3390/v6124811.

1984;81(20):6349-53.

162-69. DOI: 10.1016/j.gene.2013.03.137.

5270-76.

ma. Virology. 1959;7(1):75-91.

and Erika Durinikova

the United States of America. 1968;60(4):1288-95.

sarcoma cells in tissue culture. Virology. 1958;6(3):669-88.

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia

[1] Sambrook J, Westphal H, Srinivasan PR, Dulbecco R: The integrated state of viral DNA in SV40-transformed cells. Proceedings of the National Academy of Sciences of

[2] Zinder ND, Lederberg J: Genetic exchange in Salmonella. Journal of bacteriology.

[3] Temin HM, Rubin H: Characteristics of an assay for Rous sarcoma virus and Rous

[4] Rubin H, Temin HM: A radiological study of cell-virus interaction in the Rous sarco‐

[5] Temin HM: Mixed infection with two types of Rous sarcoma virus. Virology.

[6] Temin HM: The Participation of DNA in Rous sarcoma virus production. Virology.

[7] Suerth JD, Labenski V, Schambach A: Alpharetroviral vectors: from a cancer-causing agent to a useful tool for human gene therapy. Viruses. 2014;6(12):4811-38. DOI:

[8] Cone RD, Mulligan RC: High-efficiency gene transfer into mammalian cells: genera‐ tion of helper-free recombinant retrovirus with broad mammalian host range. Pro‐ ceedings of the National Academy of Sciences of the United States of America.

[9] Mann R, Mulligan RC, Baltimore D: Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983;33(1):153-59.

[10] Page KA, Landau NR, Littman DR: Construction and use of a human immunodefi‐ ciency virus vector for analysis of virus infectivity. Journal of virology. 1990;64(11):

[11] Wirth T, Parker N, Yla-Herttuala S: History of gene therapy. Gene. 2013;525(2):

\*Address all correspondence to: exonmigu@savba.sk


[40] Chen R: Enzyme engineering: rational redesign versus directed evolution. Trends in Biotechnology. 2001;19(1):13-14.

[26] Baum C, Schambach A, Bohne J, Galla M: Retrovirus vectors: toward the plentivirus? Molecular Therapy. 2006;13(6):1050-63. DOI: 10.1016/j.ymthe.2006.03.007.

[27] Maetzig T, Galla M, Baum C, Schambach A: Gammaretroviral vectors: biology, tech‐ nology and application. Viruses. 2011;3(6):677-713. DOI: 10.3390/v3060677.

[28] Durinikova E, Kucerova L, Matuskova M: Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene ther‐

[29] Bank A, Markowitz DG, Goff SP: Retroviral packaging cell lines and process of using

[30] Tailor CS, Nouri A, Lee CG, Kozak C, Kabat D: Cloning and characterization of a cell surface receptor for xenotropic and polytropic murine leukemia viruses. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(3):

[31] Durand S, Cimarelli A: The inside out of lentiviral vectors. Viruses. 2011;3(2):132-59.

[32] Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al.: In vivo gene de‐ livery and stable transduction of nondividing cells by a lentiviral vector. Science.

[33] Yu SF, von Ruden T, Kantoff PW, Garber C, Seiberg M, Ruther U, et al.: Self-inacti‐ vating retroviral vectors designed for transfer of whole genes into mammalian cells. Proceedings of the National Academy of Sciences of the United States of America.

[34] Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracy‐ cline-responsive promoters. Proceedings of the National Academy of Sciences of the

[35] Russell SJ, Peng KW, Bell JC: Oncolytic virotherapy. Nature Biotechnology.

[36] Tai CK, Kasahara N: Replication-competent retrovirus vectors for cancer gene thera‐ py. Frontiers in Bioscience : A Journal and Virtual Library. 2008;13:3083-95.

[37] Schambach A, Bohne J, Chandra S, Will E, Margison GP, Williams DA, et al.: Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methyl‐ guanine-DNA methyltransferase in hematopoietic cells. Molecular Therapy.

[38] Gabriel R, Schmidt M, von Kalle C: Integration of retroviral vectors. Current Opinion

[39] Yi Y, Noh MJ, Lee KH: Current advances in retroviral gene therapy. Current Gene

apy. Acta virologica. 2014;58(1):1-13.

same. Google Patents; 1994.

DOI: 10.3390/v3020132.

1996;272(5259):263-67.

1986;83(10):3194-208.

United States of America. 1992;89(12):5547-51.

2006;13(2):391-400. DOI: 10.1016/j.ymthe.2005.08.012.

in Immunology. 2012;24(5):592-97. DOI: 10.1016/j.coi.2012.08.006.

2012;30(7):658-70. DOI: 10.1038/nbt.2287.

Therapy. 2011;11(3):218-28.

927-32.

160 Advances in Molecular Retrovirology


[64] Scartozzi M, Maccaroni E, Giampieri R, Pistelli M, Bittoni A, Del Prete M, et al.: 5- Fluorouracil pharmacogenomics: still rocking after all these years? Pharmacogenom‐ ics. 2011;12(2):251-65. DOI: 10.2217/pgs.10.167.

[52] Murakami Y, Ikeda Y, Yonemitsu Y, Miyazaki M, Inoue M, Hasegawa M, et al.: In‐ hibition of choroidal neovascularization via brief subretinal exposure to a newly de‐ veloped lentiviral vector pseudotyped with Sendai viral envelope proteins. Human

[53] McFarland TJ, Zhang Y, Appukuttan B, Stout JT: Gene therapy for proliferative ocu‐ lar diseases. Expert Opinion on Biological Therapy. 2004;4(7):1053-58. DOI:

[54] Perdoni C, Osborn MJ, Tolar J: Gene editing toward the use of autologous therapies in recessive dystrophic epidermolysis bullosa. Translational Research: The Journal of Laboratory and Clinical Medicine. 2015;S1931-5244(15)00175-9. DOI: 10.1016/j.trsl.

[55] Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A, et al.: Correc‐ tion of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nature Medicine. 2006;12(12):1397-402. DOI: 10.1038/nm1504.

[56] Piccolo P, Brunetti-Pierri N: Gene therapy for inherited diseases of liver metabolism.

[57] Grossman M, Raper SE, Kozarsky K, Stein EA, Engelhardt JF, Muller D, et al.: Suc‐ cessful ex vivo gene therapy directed to liver in a patient with familial hypercholes‐

[58] Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al.: Lentiviral hemato‐ poietic stem cell gene therapy benefits metachromatic leukodystrophy. Science.

[59] Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, et al.: Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adreno‐

leukodystrophy. Science. 2009;326(5954):818-23. DOI: 10.1126/science.1171242.

pects of Medicine. 2007;28(1):4-41. DOI: 10.1016/j.mam.2006.12.001.

[60] Portsmouth D, Hlavaty J, Renner M: Suicide genes for cancer therapy. Molecular As‐

[61] Adachi M, Sampath J, Lan LB, Sun D, Hargrove P, Flatley R, et al.: Expression of MRP4 confers resistance to ganciclovir and compromises bystander cell killing. The Journal of Biological Chemistry. 2002;277(41):38998-9004. DOI: 10.1074/

[62] Nicholas TW, Read SB, Burrows FJ, Kruse CA: Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to im‐ prove gap junctions and bystander effects. Histology and Histopathology. 2003;18(2):

[63] Kievit E, Nyati MK, Ng E, Stegman LD, Parsels J, Ross BD, et al.: Yeast cytosine de‐ aminase improves radiosensitization and bystander effect by 5-fluorocytosine of hu‐

man colorectal cancer xenografts. Cancer Research. 2000;60(23):6649-55.

Human gene therapy. 2015;26(4):186-92. DOI: 10.1089/hum.2015.029.

terolaemia. Nature Genetics. 1994;6(4):335-41. DOI: 10.1038/ng0494-335.

2013;341(6148):1233158. DOI: 10.1126/science.1233158.

Gene Therapy. 2010;21(2):199-209. DOI: 10.1089/hum.2009.102.

10.1517/14712598.4.7.1053.

2015.05.008.

162 Advances in Molecular Retrovirology

jbc.M203262200.

495-507.


hibit human prostate tumor growth. Molecular Therapy. 2010;18(1):223-31. DOI: 10.1038/mt.2009.237.

[90] Abrate A, Buono R, Canu T, Esposito A, Del Maschio A, Luciano R, et al.: Mesenchy‐ mal stem cells expressing therapeutic genes induce autochthonous prostate tumour regression. European Journal of Cancer. 2014;50(14):2478-88. DOI: 10.1016/j.ejca. 2014.06.014.

[76] Klopp AH, Gupta A, Spaeth E, Andreeff M, Marini F, 3rd: Concise review: dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tu‐

[77] Kucerova L, Matuskova M, Hlubinova K, Altanerova V, Altaner C: Tumor cell be‐ haviour modulation by mesenchymal stromal cells. Molecular Cancer. 2010;9:129.

[78] Junttila MR, de Sauvage FJ: Influence of tumour micro-environment heterogeneity on therapeutic response. Nature. 2013;501(7467):346-54. DOI: 10.1038/nature12626. [79] Cihova M, Altanerova V, Altaner C: Stem cell based cancer gene therapy. Molecular

[80] Osaka M, Honmou O, Murakami T, Nonaka T, Houkin K, Hamada H, et al.: Intrave‐ nous administration of mesenchymal stem cells derived from bone marrow after con‐ tusive spinal cord injury improves functional outcome. Brain Research.

[81] Akiyama Y, Radtke C, Honmou O, Kocsis JD: Remyelination of the spinal cord fol‐ lowing intravenous delivery of bone marrow cells. Glia. 2002;39(3):229-36. DOI:

[82] Bexell D, Svensson A, Bengzon J: Stem cell-based therapy for malignant glioma. Can‐ cer Treatment Reviews. 2013;39(4):358-65. DOI: 10.1016/j.ctrv.2012.06.006.

[83] Morizono K, De Ugarte DA, Zhu M, Zuk P, Elbarbary A, Ashjian P, et al.: Multiline‐ age cells from adipose tissue as gene delivery vehicles. Human Gene Therapy.

[84] Keung EZ, Nelson PJ, Conrad C: Concise review: genetically engineered stem cell therapy targeting angiogenesis and tumor stroma in gastrointestinal malignancy.

[85] Kucerova L, Poturnajova M, Tyciakova S, Matuskova M: Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cyto‐ sine deaminase. Stem Cell Research. 2012;8(2):247-58. DOI: 10.1016/j.scr.2011.11.006.

[86] Dwyer RM, Khan S, Barry FP, O'Brien T, Kerin MJ: Advances in mesenchymal stem cell-mediated gene therapy for cancer. Stem Cell Research & Therapy. 2010;1(3):25.

[87] Zhang J, Temin HM: Rate and mechanism of nonhomologous recombination during

[88] Kucerova L, Altanerova V, Matuskova M, Tyciakova S, Altaner C: Adipose tissue-de‐ rived human mesenchymal stem cells mediated prodrug cancer gene therapy. Can‐

[89] Cavarretta IT, Altanerova V, Matuskova M, Kucerova L, Culig Z, Altaner C: Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme in‐

a single cycle of retroviral replication. Science. 1993;259(5092):234-38.

cer Research. 2007;67(13):6304-13. DOI: 10.1158/0008-5472.CAN-06-4024.

mor growth? Stem Cells. 2011;29(1):11-19. DOI: 10.1002/stem.559.

Pharmaceutics. 2011;8(5):1480-87. DOI: 10.1021/mp200151a.

2010;1343:226-35. DOI: 10.1016/j.brainres.2010.05.011.

2003;14(1):59-66. DOI: 10.1089/10430340360464714.

Stem Cells. 2013;31(2):227-35. DOI: 10.1002/stem.1269.

DOI: 10.1186/1476-4598-9-129.

164 Advances in Molecular Retrovirology

10.1002/glia.10102.

DOI: 10.1186/scrt25.


HSV-Tk gene on U-87-driven brain tumor. PloS One. 2015;10(6):e0128922. DOI: 10.1371/journal.pone.0128922.


## **Endogenous Retroviruses**

HSV-Tk gene on U-87-driven brain tumor. PloS One. 2015;10(6):e0128922. DOI:

[101] Matuskova M, Kozovska Z, Toro L, Durinikova E, Tyciakova S, Cierna Z, et al.: Com‐ bined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases. Journal of Experi‐ mental & Clinical Cancer Research: CR. 2015;34:33. DOI: 10.1186/s13046-015-0149-2.

[102] Tyciakova S, Matuskova M, Bohovic R, Polakova K, Toro L, Skolekova S, et al.: Ge‐ netically engineered mesenchymal stromal cells producing TNFalpha have tumour suppressing effect on human melanoma xenograft. The Journal of Gene Medicine.

[103] Yan C, Yang M, Li Z, Li S, Hu X, Fan D, et al.: Suppression of orthotopically implant‐ ed hepatocarcinoma in mice by umbilical cord-derived mesenchymal stem cells with sTRAIL gene expression driven by AFP promoter. Biomaterials. 2014;35(9):3035-43.

[104] Deng Q, Zhang Z, Feng X, Li T, Liu N, Lai J, et al.: TRAIL-secreting mesenchymal stem cells promote apoptosis in heat-shock-treated liver cancer cells and inhibit tu‐ mor growth in nude mice. Gene Therapy. 2014;21(3):317-27. DOI: 10.1038/gt.2013.88.

[105] Hong SH, Lee HJ, An J, Lim I, Borlongan C, Aboody KS, et al.: Human neural stem cells expressing carboxyl esterase target and inhibit tumor growth of lung cancer brain metastases. Cancer Gene Therapy. 2013;20(12):678-82. DOI: 10.1038/cgt.2013.69.

[106] Sugrue T, Lowndes NF, Ceredig R: Mesenchymal stromal cells: radio-resistant mem‐ bers of the bone marrow. Immunology and Cell Biology. 2013;91(1):5-11. DOI:

[107] Altaner C, Altanerova V: Stem cell based glioblastoma gene therapy. Neoplasma.

[108] Matuskova M, Baranovicova L, Kozovska Z, Durinikova E, Pastorakova A, Hunako‐ va L, et al.: Intrinsic properties of tumour cells have a key impact on the bystander effect mediated by genetically engineered mesenchymal stromal cells. The Journal of

[109] Lee JY, Lee DH, Kim HA, Choi SA, Lee HJ, Park CK, et al.: Double suicide gene ther‐ apy using human neural stem cells against glioblastoma: double safety measures. Journal of Neuro-Oncology. 2014;116(1):49-57. DOI: 10.1007/s11060-013-1264-6. [110] Niess H, von Einem JC, Thomas MN, Michl M, Angele MK, Huss R, et al.: Treatment of advanced gastrointestinal tumors with genetically modified autologous mesen‐ chymal stromal cells (TREAT-ME1): study protocol of a phase I/II clinical trial. BMC

10.1371/journal.pone.0128922.

166 Advances in Molecular Retrovirology

2015;17(1-2):54-67. DOI: 10.1002/jgm.2823.

DOI: 10.1016/j.biomaterials.2013.12.037.

2012;59(6):756-60. DOI: 10.4149/neo\_2012\_95.

Gene Medicine. 2012;14(12):776-87. DOI: 10.1002/jgm.2684.

Cancer. 2015;15(1):237. DOI: 10.1186/s12885-015-1241-x.

10.1038/icb.2012.61.

### **HERVs in Multiple Sclerosis — From Insertion to Therapy**

Belén de la Hera and Elena Urcelay

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61726

#### **Abstract**

Genome-wide association studies (GWAS) have not been able to completely elucidate the genetic background of complex diseases. Part of it could lie in repetitive sequences not studied in the GWAS, as those corresponding to Human Endogenous Retroviruses (HERVs). In the present work, we aim to review the potential role of HERVs in the etiology of autoimmune diseases, especially in multiple sclerosis (MS); their potential pathogenic role and their putative consideration as a good target for new treatments. For this purpose, we carried out an in-depth literature review on HERVs, and we integrated our previous findings about HERV-W, HERV-K18, and HERV-Fc1 and MS susceptibility. The study was carried out by a systematic search from electronic databases using the keywords "HERV," "Multiple sclerosis," "HERV-W," "MSRV," "HERV-K," "HERV-Fc1," and "GNbAC1."

**Keywords:** Multiple sclerosis, HERV, MSRV, GNbAC1

#### **1. Introduction**

#### **1.1. HERVs**

The endogenous retroviruses (ERVs) could be defined as "genetic parasites" of vertebrates [1], given that their origin is very different from the one displayed by the rest of the genome. Their existence in the genome of mammals is only known since 1970 [2], although, they resulted from ancestral infections by exogenous retroviruses millions of years ago. During an infection, the exogenous retroviruses are able to integrate one copy of their genome (provirus) into the genome of the host. Thus, they can stay permanently associated with the host and be trans‐ mitted horizontally by the creation of new virions (the typical spread of an infectious virus). Only when they infect a germ line cell, the integrated DNA can become part of the gene pool

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and be transmitted in a Mendelian fashion like ERVs [1, 3-5], as shown in Figure 1. Those who are present in the human genome are named human endogenous retroviruses (HERVs).

The endogenization process profoundly impacts on the survival and evolution of the virus and the host. It results from the balance achieved between the immune surveillance and the virus virulence [6]. In this way, the HERVs must surpass the host's antiviral defense mecha‐ nisms and infect the germ cells without causing a cytotoxicity that would prevent persistence in the progeny of the host [6]. Furthermore, from this moment on, all host cells are carriers of an integrated provirus [6].

**Figure 1. The endogenization process**: once the retrovirus infects a germ line cell, vertical transmission in a Mendelian fashion occurs.

The retroviral insertion is aleatory, in the sense that no specific sites for retroviral integration exist in the host genome. Nonetheless, due to the epigenetic chromatin packaging, integrated HERVs elements are more commonly found within the transcriptionally active genome [6]. Currently, HERVs comprise nearly 8% of the human genome [7], distributed in approximately 31 independently acquired multigene families [8]. Even though no standard nomenclature has been defined for HERVs, they have been classified based on their homology with different groups of exogenous retroviruses. They are grouped as class I, class II, or class III retroviruses considering their homology with Gamma and Epsilon retroviruses, Betaretrovirus or Spuma‐ virus, respectively [9, 10]. The family name is usually given by "HERV" followed by a oneletter amino acid code that corresponds to the tRNA specific of the site used to initiate reverse transcription [10]; consequently, the HERV-W family would use a tryptophan.

As mentioned, HERVs have a similar structure to proviruses of infectious retroviruses, with three principal genes, *gag*, *pol*, and *env,* flanked by two long terminal repeats (LTRs) [6]. The *gag* gene codes for the viral assembly proteins, including the nucleocapsid, matrix, and capsid proteins. The *pol* gene codes for the viral replication proteins, yielding the reverse transcriptase, protease, ribonuclease, and integrase proteins. Finally, the *env* gene codes for a viral glyco‐ protein, with both a surface and a transmembrane subunit. However, important changes are observed in the HERVs expression compared to that of exogenous retroviruses. Most HERVs encode incomplete proteins and accumulate mutations and recombinations. Furthermore, most HERVs with functional LTRs remain in a latent state under homeostatic conditions, owing to the epigenetic silencing of the provirus in heterochromatin [11]. Exceptionally, specific HERVs have been selected during evolution, provided that their biological functions could be beneficial for the host. In these cases, HERVs suffer a "domestication," meaning that a foreign gene can be used for cellular functions of the host [12]. In this group, we find proteins like Syncytin-1 from the HERV-W family, and Syncytin-2 from the HERV-FRD family [13]. These highly fusogenic envelope proteins are necessary to allow the formation of the placental syncytiotrophoblast layer; furthermore, they could be involved in the immune tolerance to the fetus [13].

In addition to these "domestic HERVs," several studies show reactivation of HERVs under pathologic conditions, such as different types of cancer [14-20]; autoimmune diseases includ‐ ing multiple sclerosis (MS) [21-37], rheumatoid arthritis (RA) [38], psoriasis [39], or systemic lupus erythematosus (SLE) [40]; and other diseases like schizophrenia [41, 42]. Nonetheless, we do not know whether their reactivation or increased expression is a causal effect, or conversely, is an underlying consequence of the disease.

#### **1.2. Potential expression mechanisms of HERVs**

Many factors can interfere or modulate the expression of HERVs, such as recombination events between two or more replication-defective HERVs [43, 44], infectious agents like Human herpesvirus 6 (HHV6) [34, 45] and Epstein–Barr virus (EBV) [46, 47], several transcription factors [31, 48], and the epigenomic context of the HERVs [6, 49, 50].

**•** Recombination events

and be transmitted in a Mendelian fashion like ERVs [1, 3-5], as shown in Figure 1. Those who are present in the human genome are named human endogenous retroviruses (HERVs).

The endogenization process profoundly impacts on the survival and evolution of the virus and the host. It results from the balance achieved between the immune surveillance and the virus virulence [6]. In this way, the HERVs must surpass the host's antiviral defense mecha‐ nisms and infect the germ cells without causing a cytotoxicity that would prevent persistence in the progeny of the host [6]. Furthermore, from this moment on, all host cells are carriers of

**Figure 1. The endogenization process**: once the retrovirus infects a germ line cell, vertical transmission in a Mendelian

The retroviral insertion is aleatory, in the sense that no specific sites for retroviral integration exist in the host genome. Nonetheless, due to the epigenetic chromatin packaging, integrated HERVs elements are more commonly found within the transcriptionally active genome [6]. Currently, HERVs comprise nearly 8% of the human genome [7], distributed in approximately 31 independently acquired multigene families [8]. Even though no standard nomenclature has been defined for HERVs, they have been classified based on their homology with different groups of exogenous retroviruses. They are grouped as class I, class II, or class III retroviruses considering their homology with Gamma and Epsilon retroviruses, Betaretrovirus or Spuma‐ virus, respectively [9, 10]. The family name is usually given by "HERV" followed by a oneletter amino acid code that corresponds to the tRNA specific of the site used to initiate reverse

As mentioned, HERVs have a similar structure to proviruses of infectious retroviruses, with three principal genes, *gag*, *pol*, and *env,* flanked by two long terminal repeats (LTRs) [6]. The *gag* gene codes for the viral assembly proteins, including the nucleocapsid, matrix, and capsid

transcription [10]; consequently, the HERV-W family would use a tryptophan.

an integrated provirus [6].

170 Advances in Molecular Retrovirology

fashion occurs.

Two or more replication-defective HERVs can restore their own defects through recombination events, resulting in a replication-competent retrovirus [5]. Even though this is an infrequent event, a study in mice points to a significantly increased frequency in specific immune deficiencies [44]. Furthermore, it has been demonstrated that recombination between three HERV-K defective proviruses is possible, leading to an infectious retrovirus [5, 43].

**•** Infectious agents

A putative explanation about the preferential expression of HERVs found in human brain samples could be the tropism of specific viruses and bacteria to the central nervous system (CNS). Neurotropic agents like herpesvirus [29, 51], Toxoplasma gondii [52], or certain strands of influenza virus [53] are able to cross the hematoencephalic barrier into the CNS. Usually, they are intercepted by cerebral macrophages leading to an abortive infection, but their transient presence in the CNS could activate the HERVs expression as a consequence of their immediate-early (IE) genes expression [4]. The expression of the IE genes of herpes simplex virus type 1 (HSV-1) and its interaction with the transcription factor binding sites situated in the U3 region of the LTR, such as AP-1 [54] and Oct-1 [55], lead to an activation of transcription in HERV-K and HERV-W families.

The herpesviruses are one of the best candidates: they may be neurotropic, remain latent, and can be reactivated. Furthermore, the expression of the Env epitopes in the surface of B cells and monocytes could be a consequence of the interaction between HERVs and herpesviruses [25]. Thus, the herpesviruses could play a dual role in neurodegenerative diseases, acting as pathological entities *per se* and as inducers of HERVs [6].

**•** Transcription factors

An important component of the antiviral innate immunity is the regulation of the expression and replication of HERVs by different transcription factors [48]. In HERV-W and HERV-K elements, both families previously related to multiple sclerosis (MS), different binding sites for transcription factors such as NF-Kβ [31, 48] are located in their promoter regions and could drive an increased expression of HERVs during inflammation.

**•** Epigenomic context

The chromatin state as well as the methylation state of GpC islands within the HERV promoter and regulatory regions seem to be crucial factors in the control of HERVs expression [49, 50]. Both play an important role as a part of the defense system against the potential effects of inserted sequences. Previously published studies describe how proviruses and solitary LTRs are densely methylated under physiological conditions, but hypomethylated in placenta [49, 50]. Thus, DNA hypomethylation, as observed in certain types of cancer, could allow reacti‐ vation of retroelements. In MS, HERVs have been described as susceptible elements to undergo epigenetic modifications, mainly due to modifications in the methylation state, resulting in activation of their expression and, consequently, inappropriate activation of the immune system.

#### **1.3. Pathogenic mechanisms of HERVs**

Even though most inserted copies in the human genome are defective copies, some HERVs could maintain the potential to cause or contribute to disease by different mechanisms [5]. As mentioned, HERVs may alter cellular functions by two ways, either acting as a genetic element or as a viral pathogen [6].

**•** Gene disruption

HERVs, like transposons, are able to experience transposition, recombination, and integration cycles. Some HERVs families include a high number of copies in the genome. It is believed that these families have been spread around the genome through the reintegration of a provirus. However, each new integration process increases the risk of a harmful insertion. They can disrupt genes present in their integration sites, for example, HERV-K integrations have been identified into tumor suppressor genes like *BRCA2* and into the repair *XRCC1* gene [6, 56].

**•** Modulation of gene expression

Some HERVs conserve regulatory sequences that can operate as functional promoters, enhancers, or polyadenylation signals, so they could change the expression of adjacent or distal genes [4]. They can also form part of regulatory RNAs: microRNAs (miRNA), small interfering RNAs (siRNA), and long intergenic noncoding RNAs (lincRNAs), contributing to the complex regulatory network of gene expression [5]. Furthermore, HERVs integrated into introns can provide alternative transcription start and termination sites [5].

**•** Pattern recognition receptors (PRRs)

virus type 1 (HSV-1) and its interaction with the transcription factor binding sites situated in the U3 region of the LTR, such as AP-1 [54] and Oct-1 [55], lead to an activation of transcription

The herpesviruses are one of the best candidates: they may be neurotropic, remain latent, and can be reactivated. Furthermore, the expression of the Env epitopes in the surface of B cells and monocytes could be a consequence of the interaction between HERVs and herpesviruses [25]. Thus, the herpesviruses could play a dual role in neurodegenerative diseases, acting as

An important component of the antiviral innate immunity is the regulation of the expression and replication of HERVs by different transcription factors [48]. In HERV-W and HERV-K elements, both families previously related to multiple sclerosis (MS), different binding sites for transcription factors such as NF-Kβ [31, 48] are located in their promoter regions and could

The chromatin state as well as the methylation state of GpC islands within the HERV promoter and regulatory regions seem to be crucial factors in the control of HERVs expression [49, 50]. Both play an important role as a part of the defense system against the potential effects of inserted sequences. Previously published studies describe how proviruses and solitary LTRs are densely methylated under physiological conditions, but hypomethylated in placenta [49, 50]. Thus, DNA hypomethylation, as observed in certain types of cancer, could allow reacti‐ vation of retroelements. In MS, HERVs have been described as susceptible elements to undergo epigenetic modifications, mainly due to modifications in the methylation state, resulting in activation of their expression and, consequently, inappropriate activation of the immune

Even though most inserted copies in the human genome are defective copies, some HERVs could maintain the potential to cause or contribute to disease by different mechanisms [5]. As mentioned, HERVs may alter cellular functions by two ways, either acting as a genetic element

HERVs, like transposons, are able to experience transposition, recombination, and integration cycles. Some HERVs families include a high number of copies in the genome. It is believed that these families have been spread around the genome through the reintegration of a provirus. However, each new integration process increases the risk of a harmful insertion. They can disrupt genes present in their integration sites, for example, HERV-K integrations have been identified into tumor suppressor genes like *BRCA2* and into the repair *XRCC1* gene [6, 56].

in HERV-K and HERV-W families.

**•** Transcription factors

172 Advances in Molecular Retrovirology

**•** Epigenomic context

**1.3. Pathogenic mechanisms of HERVs**

or as a viral pathogen [6].

**•** Modulation of gene expression

**•** Gene disruption

system.

pathological entities *per se* and as inducers of HERVs [6].

drive an increased expression of HERVs during inflammation.

The HERVs expression products, both nucleic acids and proteins, can modulate immune responses. They have the potential to interact with components involved in the immune innate response and to activate proinflammatory signaling pathways [57, 58]. Therefore, certain HERVs proteins could directly interact with specific toll-like receptors (TLRs), for example with TLR4, resulting in the production of TNFα and proinflammatory cytokines [58-60]. The nucleic acids derived from HERVs may also activate cytosolic PRRs; in this way, both an increased expression of RNA and the presence of cDNA in a nonfamilial compartment like the cytosol could activate PRRs [60]. Nonetheless, the human being has coevolved with endoge‐ nous retroelements and this could have shaped the sensibility of DNA sensors of the innate immune system, leading to an increased cDNA detection threshold to avoid an immune response against them. The cDNA levels are restrained by the action of gene products like Trex1 or SAMHD1 [60] and a loss-of-function mutation in these enzymes could result in the cDNA accumulation and the consequent sensors activation. This process would lead to a chronic immune response with release of pathogenic type I IFN and inflammatory mediators, similar to those observed in autoimmune diseases [60].

**•** Viral proteins: molecular mimicry, superantigen activity, or immunosuppressive proteins

HERVs proteins hold epitopes to B and T cells and molecular mimicry between viral proteins and certain autoantigens may exist, resulting in an autoimmune response. Moreover, some HERVs sequences are able to encode for superantigens. Superantigens combine with MHC class-II molecules to form ligands that stimulate T cells [61], and this may end in an abnormal activation of autoreactive T lymphocytes [62].

Alternatively, evidences exist of the immunosuppressive activity of certain HERVs Env proteins [63, 64]. This activity is reminiscent of their exogenous antecessors, which in this way increased the viability of the virions in the host. This capacity has suffered an adaptation process, and nowadays it might be implicated in the materno-fetal tolerance and could also prevent the immune response to exogen pathogens and tumors [60].

**•** Retroviral help for B cells

HERVs can also help B cells to quickly produce antibodies directed against pathogenic antigens [65]. The bacterial polysaccharide antigens and the carbohydrates linked to viral glycoproteins have the ability to stimulate B cells in the absence of T-cell help. These antigens are called thymus-independent antigens (TI), and they can be classified into two types: TI-1 or TI-2 antigens. TI-2 antigens cause extensive cross-linking of the BCR, leading to a quick differen‐ tiation of B cells into plasma cells. Finally, these plasma cells secrete protective antibodies, IgM and IgG [66]. However, the mechanism by which the TI-2 antigens activate B cells in the marginal zone without the help of T cells still remains poorly understood. It has been recently described that the cross-linking of B cells activates a signaling cascade, including the Bruton Tyrosine Kinase and the nuclear transcription factor NF-Kβ, allowing transcription of endog‐ enous retroviral DNA [66]. The retroviral RNA may activate B cells by two complementary but different pathways: first, it could activate the retinoic-acid-inducible gene 1 receptor (RIG-1), resulting in a mitochondrial antiviral-signaling (MAVS); second, the RNA can be converted into DNA and can activate the cyclic GMP-AMP synthase (cGAS, cGAMP synthase). Finally, both signaling pathways would finish in the antigen-specific B-cell activation [65, 66].

#### **2. HERVs and autoimmune diseases**

HERVs represent the immunological limit between the self and the foreign. Their peculiar origin is very different from that of other genome elements, as they can share properties with infectious agents. Indeed, in case they would produce particles, these would not be so different from those originated from exogenous retroviruses. Therefore, they could activate the immune system and would induce autoimmunity [67]. As it has been previously discussed, HERVs have been associated, among other infectious or neurologic diseases, with different autoim‐ mune diseases like MS [21-37], RA [38], psoriasis [39], T1D [68], or SLE [40], as shown in Table 1. Genome-wide association studies (GWAS) showed the existence of a genetic basis shared between different autoimmune diseases, discovering new immunogenic mechanisms impli‐ cated, and HERVs could be part of these shared genetic elements.



MS, multiple sclerosis; HIV, human immunodeficiency virus; SLE, Systemic lupus erythematosus; ALS, amyotrophic lateral sclerosis; T1D, type 1 diabetes; RA, rheumatoid arthritis, HTLV-1, human T-lymphotropic virus-1; IFN, interferon; Env, envelope; OPCs, olygodendrocyte precursor cells; N/A, not applicable. Based on Douville and Nath, 2014 [6].

**Table 1.** HERVs families associated with different diseases

#### **3. HERVs and MS**

and IgG [66]. However, the mechanism by which the TI-2 antigens activate B cells in the marginal zone without the help of T cells still remains poorly understood. It has been recently described that the cross-linking of B cells activates a signaling cascade, including the Bruton Tyrosine Kinase and the nuclear transcription factor NF-Kβ, allowing transcription of endog‐ enous retroviral DNA [66]. The retroviral RNA may activate B cells by two complementary but different pathways: first, it could activate the retinoic-acid-inducible gene 1 receptor (RIG-1), resulting in a mitochondrial antiviral-signaling (MAVS); second, the RNA can be converted into DNA and can activate the cyclic GMP-AMP synthase (cGAS, cGAMP synthase). Finally, both signaling pathways would finish in the antigen-specific B-cell activation [65, 66].

HERVs represent the immunological limit between the self and the foreign. Their peculiar origin is very different from that of other genome elements, as they can share properties with infectious agents. Indeed, in case they would produce particles, these would not be so different from those originated from exogenous retroviruses. Therefore, they could activate the immune system and would induce autoimmunity [67]. As it has been previously discussed, HERVs have been associated, among other infectious or neurologic diseases, with different autoim‐ mune diseases like MS [21-37], RA [38], psoriasis [39], T1D [68], or SLE [40], as shown in Table 1. Genome-wide association studies (GWAS) showed the existence of a genetic basis shared between different autoimmune diseases, discovering new immunogenic mechanisms impli‐

**Class Family PBS Related diseases Expression mechanisms Pathogenic mechanisms**

I HERV-F Phe MS Demethylating agents Superantigen activity

<sup>I</sup> HERV-E Glu SLE Hypomethylation Immunosupressor potential of

I HERV-P Pro Cancer Unkown Unknown

Herpesviruses Transcription factors Toxoplasma gondii Influenza A virus

> Herpersviruses HTLV-1 Type 1 IFN

Pro-inflammatory Env protein Superantigen activity OPCs differentiation interference Altered glial function

recombination

Env

Superantigen activity Neoepitopes Genes disruption

N/A Genetic deletion by

**2. HERVs and autoimmune diseases**

174 Advances in Molecular Retrovirology

I HERV-W Trp

I HERV-H His

II HERV-K Lys

cated, and HERVs could be part of these shared genetic elements.

MS Schizophrenia HIV Osteoarthitis

3q13.31 microdeletion syndrome

> MS ALS HIV

MS is one of the conditions more frequently related with HERVs. It is a chronic progressive disease characterized by neuroinflammation in the CNS accompanied by demyelination, axonal damage, and progressive neurologic dysfunction [69]. It is a complex disease, origi‐ nated from the interaction of genetic, environmental, and epigenetic factors [70]. Recently, its incidence seems to be increased; at present MS affects 2.3 million people in the world [71]. However, many aspects of its pathogenesis are still poorly understood. GWAS have not completely explained the MS genetic background [72-74], albeit including the ImmunoChip Project [75] a total of 110 single nucleotide polymorphisms (SNPs) have been associated with MS susceptibility. Even considering the strongest risk factor, the HLA-DRB1\*15:01 allele, each SNP has a modest effect and all together are able to explain only 20–28% of MS heritability [75]. Part of the missing heritability could reside on HERVs, as repetitive regions were not analyzed in the GWAS. Those repetitive regions were previously considered as "junk DNA" because it was thought that they had little or no physiological role. However, nowadays we know that these sequences could play an important role in the development of autoimmune diseases, including MS.

In 1989, Perron et al. [76] described the presence of extracellular virions associated with reverse transcriptase activity in a culture of leptomeningeal cells (LM7) obtained from the cerebrospi‐ nal fluid (CSF) of an MS patient. In the beginning, it was thought that those virions could correspond to the human T-lymphotropic virus (HTLV-1) due to the similarities between the tropical spastic paraparesis (a demyelinating progressive disease) caused by HTLV-1, and MS. However, a new retroviral element called MSRV (Multiple-sclerosis-associated retrovirus) was identified, the founder of the HERV-W family [77]. This multicopy family, consisting of approximately 650 loci around the human genome [35], comprises a total of 311 inserts (more or less complete proviruses or pseudogenes) and 343 additional HERV-W LTRs [78].

Only the *env* gene mapping on chromosome 7, encoding Syncytin, presents a complete open reading frame (ORF) and has been selectively conserved [35]. The MSRV *env* sequence can be differentiated from the one corresponding to Syncytin-1 by a 12-nucleotide insertion in the transmembrane moiety. Both genes are expressed in the brain of MS patients, but the MSRVtype *env* DNA copies were found sixfold more frequently in MS patients than in healthy controls, while comparable copy numbers of Syncytin-1 were observed [79]. Furthermore, Syncytin-1 is originated from the retroviral copy inserted in chromosome 7, and the pathogenic protein MSRV-type Env could be originated from several integrations in the human genome, or it could result from recombination events between insertions in different chromosomes [80, 81]. The genomic origin of HERV-W Env remains unknown although recent works consider the copy mapping to chromosome X one plausible candidate [4, 80, 81]. This copy, located on Xq22.3, would encode for an almost complete MSRV-type protein, truncated on its N-terminal end due to the presence of a stop codon mutation at position 39 [81]. *Ex vivo*, this copy still conserves coding capacity, as it is able to produce a truncated N-terminally Env protein [80]. Furthermore, the reversion of this stop codon would lead to a complete protein with signal peptide, expressed in the cellular surface in the same way that Syncytin [80].

Recently, a genetic screening was performed by specific PCR amplification followed by High Resolution Melting (HRM) analyses of the two MSRV-like env copies which show the ORF with the highest length similarity and homology to Syncytin (1614 bp), inserted in chromosome X (1428 bp) and in chromosome 20 (1419 bp). Both chromosomal origins show similar lengths of their respective ORFs, 10% shorter than the one measured for Syncytin, and could putatively originate functional proteins. The results pointed to the insertion in chromosome X, and not the one in chromosome 20, as an origin of MSRV. One polymorphism identified in chromosome X, rs6622139\*T, was associated in women with MS susceptibility and severity [82], and it was also associated with higher MSRV-like *env* levels of expression (Mann–Whitney U test: p=0.003), while the two polymorphisms found in chromosome 20 did not show evidence of association [83].

Since it was described, several studies have associated the HERV-W family with MS: the presence of MSRV-type Env protein has been found in demyelinated acute lesions in MS patients [31], as well as an increased number of DNA copies [84] or a higher prevalence of MSRV-type RNA in serum and CSF of MS patients compared with patients suffering from other neurological diseases or healthy controls from all ethnic groups [24, 27, 28, 31, 84-86]. The MSRV presence in serum and CSF is correlated with the clinical progression, severity, and prognosis of MS [28, 46], while the absence of MSRV relates with a more stable course of the disease [28, 36]. The MSRV production is stimulated by cytokines like TNFα, IL6, and IFNγ[87], and current MS therapies like IFN-β and Natalizumab, which are able to reduce MS symptomatology, promote a diminution of MSRV virus load levels in blood [87-89].

HERV-W Env proteins, MSRV-type Env, and Syncytin have proinflammatory and superanti‐ genic properties. They can cause neuroinflammation, neurodegeneration, immune system dysregulation, and endoplasmic reticulum stress [4, 21, 22, 58, 90, 91]. Their pathogenicity has been studied *in vitro* using different types of cell cultures and *in vivo* using a humanized Severe Combined Immunodeficiency Disease (SCID) animal model, showing neurotoxic effects in both settings [22, 92] and a reduced capacity of olygodendrocyte progenitor cells (OPC) differentiation, interfering in the remyelination process [57]. A recent study clarifies the possible pathogenic mechanisms of MSRV. In a human model of BBB, the endothelial cell line HCMEC/D3, they show that MRSV-type Env interacts with TLR4 and induces a dosedependent overexpression of ICAM1, as well as an induced IL6 and IL8 production; while the Env protein derived from Syncytin-1 did not show these effects [59]. Furthermore, they also described that the MSRV-type Env presence significantly stimulates the adhesion and migration of activated immune cells through the layer of endothelial cells. These results support the hypothesis that MSRV can be involved in MS pathogenesis, as well as in other chronic inflammatory diseases, at least in the maintenance of the underlying inflammatory condition [59]. Table 2 reflects the possible pathogenic mechanisms described for MSRV.


APC, antigen presenting cell; OPC, olygodendrocyte precursor cell; TCR, T-cell receptor; TLR4, toll-like receptor 4

**Table 2.** Known pathogenic mechanisms of MSRV

transmembrane moiety. Both genes are expressed in the brain of MS patients, but the MSRVtype *env* DNA copies were found sixfold more frequently in MS patients than in healthy controls, while comparable copy numbers of Syncytin-1 were observed [79]. Furthermore, Syncytin-1 is originated from the retroviral copy inserted in chromosome 7, and the pathogenic protein MSRV-type Env could be originated from several integrations in the human genome, or it could result from recombination events between insertions in different chromosomes [80, 81]. The genomic origin of HERV-W Env remains unknown although recent works consider the copy mapping to chromosome X one plausible candidate [4, 80, 81]. This copy, located on Xq22.3, would encode for an almost complete MSRV-type protein, truncated on its N-terminal end due to the presence of a stop codon mutation at position 39 [81]. *Ex vivo*, this copy still conserves coding capacity, as it is able to produce a truncated N-terminally Env protein [80]. Furthermore, the reversion of this stop codon would lead to a complete protein with signal

Recently, a genetic screening was performed by specific PCR amplification followed by High Resolution Melting (HRM) analyses of the two MSRV-like env copies which show the ORF with the highest length similarity and homology to Syncytin (1614 bp), inserted in chromosome X (1428 bp) and in chromosome 20 (1419 bp). Both chromosomal origins show similar lengths of their respective ORFs, 10% shorter than the one measured for Syncytin, and could putatively originate functional proteins. The results pointed to the insertion in chromosome X, and not the one in chromosome 20, as an origin of MSRV. One polymorphism identified in chromosome X, rs6622139\*T, was associated in women with MS susceptibility and severity [82], and it was also associated with higher MSRV-like *env* levels of expression (Mann–Whitney U test: p=0.003), while the two polymorphisms found in chromosome 20 did not show evidence of

Since it was described, several studies have associated the HERV-W family with MS: the presence of MSRV-type Env protein has been found in demyelinated acute lesions in MS patients [31], as well as an increased number of DNA copies [84] or a higher prevalence of MSRV-type RNA in serum and CSF of MS patients compared with patients suffering from other neurological diseases or healthy controls from all ethnic groups [24, 27, 28, 31, 84-86]. The MSRV presence in serum and CSF is correlated with the clinical progression, severity, and prognosis of MS [28, 46], while the absence of MSRV relates with a more stable course of the disease [28, 36]. The MSRV production is stimulated by cytokines like TNFα, IL6, and IFNγ[87], and current MS therapies like IFN-β and Natalizumab, which are able to reduce MS

symptomatology, promote a diminution of MSRV virus load levels in blood [87-89].

HERV-W Env proteins, MSRV-type Env, and Syncytin have proinflammatory and superanti‐ genic properties. They can cause neuroinflammation, neurodegeneration, immune system dysregulation, and endoplasmic reticulum stress [4, 21, 22, 58, 90, 91]. Their pathogenicity has been studied *in vitro* using different types of cell cultures and *in vivo* using a humanized Severe Combined Immunodeficiency Disease (SCID) animal model, showing neurotoxic effects in both settings [22, 92] and a reduced capacity of olygodendrocyte progenitor cells (OPC) differentiation, interfering in the remyelination process [57]. A recent study clarifies the possible pathogenic mechanisms of MSRV. In a human model of BBB, the endothelial cell line

peptide, expressed in the cellular surface in the same way that Syncytin [80].

association [83].

176 Advances in Molecular Retrovirology

Even though the HERV-W family is one of the HERV families more related to MS, other families like HERV-K18 [37, 93] or HERV-Fc1 [29, 94, 95] have also been associated with MS susceptibility.

HERV-K is a multicopy family including approximately 332 copies dispersed through the human genome. It is the only known retroviral element that codes for all the structural and enzymatic proteins (Gag, Prt, Pol), as well as for the Env protein and for the accessory Rec protein [96]. This family has been related with different autoimmune diseases as MS [37], type-1diabetes (T1D) [68], or juvenile rheumatoid arthritis [38]; and different cancer types [14-17]. One specific member of this family, HERV-K18, has been associated with MS suscept‐ ibility and its expression is induced by herpesvirus [97, 98] and by EBV [99-102], both viruses previously proposed as potential environmental factors involved in MS development [45, 51, 97, 103-108]. Three different variants of the HERV-K18 copy mapping to chromosome 1 [37] have been described. They conform haplotypes within the first intron of CD48 that can be defined by two SNPs (18.1 SNP1\*A/SNP2\*A, 18.2 SNP1\*G/SNP2\*G, 18.3 SNP1\*A/SNP2\*G), all of them coding for an Env protein with superantigenic properties. However, only one of these variants (18.3) has been associated with a higher risk to MS [37] and with an overall higher susceptibility to autoimmune diseases, as described by a meta-analysis including a total of 2656 patients and 2016 controls [93].

Considering the HERV-Fc family, a total of 6 HERV-Fc elements and 11 LTRs have been identified across the human genome. Among them, only two elements correspond to a complete HERV-Fc provirus (Fc1env and Fc2master) [109]. Related to MS, it has been observed that the HERV-Fc1 RNA levels were significantly increased in the plasma of patients suffering from active MS, compared to nonactive MS or controls [30]. The HERV-Fc1 is an unusual provirus, because it includes a single copy in the genome, located on Xq21.33. Furthermore, it is a recent acquisition for the genome, only present in humans, chimpanzees, and gorillas [109]. Nexo et al. [94] were the first to describe that rs391745, located in the promoter region of HERV-Fc1, was associated with MS susceptibility in Danish cohorts and, then, a replication study was performed with a Norwegian cohort. The latter study also detailed that the association was only observed in the nonprimary-progressive MS forms [29], results validated in further studies [95]. Regarding the HERV-Fc1 expression mechanisms, it has been observed that the transcriptional expression levels of HERV-Fc1 RNA sequences are negatively correlated with the methylation levels of CpG islands on the 5' LTR region and, therefore, a higher HERV-Fc1 expression involves DNA demethylation [11, 110].

#### **4. HERVs as future treatment options**

An increased expression of HERVs in several autoimmune diseases [21-40, 68] and different types of cancer [14-20], along with the decreased expression levels observed in successfully treated patients with immunomodulatory therapies [88, 89] or chemotherapy [111] point to the potential pathogenic role of HERVs and their putative consideration as a good target for new treatments.

A humanized monoclonal antibody anti-Env-SU MSRV/HERV-W, GNbAC1, has been studied as a putative MS treatment due to its potential neuroprotector effects [112-115]. The results of a phase IIa clinical trial [114] show that the GNbAC1 treatment blocks the transcription of proinflammatory genes mediated by Env, prevents the formation of nitrosantine, and restores OPC differentiation. Furthermore, GNbAC1 has advantages compared to other MS treatments, because the patients retain all their immune capacity. This treatment has also been studied in other diseases like diabetes and schizophrenia.

The proteins encoded by HERV-K *env* have been proposed as therapeutic targets for different types of cancer, due to the fact that a general hypomethylation of HERVs sequences has been observed, as well as an increased expression of Np9 and Rec proteins originating from HERV-K in different cancer cells [116]. Both proteins bind to the PLZF protein, a transcriptional repressor of the C-MYC proto-oncogen. The inflammation and the deregulation of protooncogen signaling caused by the HERV-K protein results in a protumorogenic microenviron‐ ment, which favors cell proliferation and metastasis [116]. The use of monoclonal antibodies against HERV-K Env protein inhibits tumoral growth and induces apoptosis in breast cancer cells *in vitro* [116]; therefore, it could be considered a good candidate as a therapy used together with other cancer treatments.

In addition to autoimmune diseases and cancer, the human immunodeficiency virus (HIV) has been also related to HERVs, particularly with the HERV-K family, raising the issue of potential beneficial effects of a therapy directed against HERVs in AIDS. Some studies report an increased expression of HERV-K provirus in HIV patients compared to controls [117, 118] and show that the immune responses against HERV-K decrease the HIV-1 viral load. *In vitro*, the use of an antibody directed against the HERV-K transmembrane protein (HML-2), HA-137, was able to eliminate the cells that displayed the antigen in their surface. This was carried out by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism by natural killer (NK) cells. It has been described that the HIV-infected cells display this membrane antigen in their surface [119]; therefore, they would be potential targets of the antibody. The possibility of finding a target epitope different from those of the HIV virus could open up opportunities to the development of vaccines against this disease; a field that has been very limited due to the high rate of mutation of the HIV [119].

#### **5. Conclusion**

Considering the HERV-Fc family, a total of 6 HERV-Fc elements and 11 LTRs have been identified across the human genome. Among them, only two elements correspond to a complete HERV-Fc provirus (Fc1env and Fc2master) [109]. Related to MS, it has been observed that the HERV-Fc1 RNA levels were significantly increased in the plasma of patients suffering from active MS, compared to nonactive MS or controls [30]. The HERV-Fc1 is an unusual provirus, because it includes a single copy in the genome, located on Xq21.33. Furthermore, it is a recent acquisition for the genome, only present in humans, chimpanzees, and gorillas [109]. Nexo et al. [94] were the first to describe that rs391745, located in the promoter region of HERV-Fc1, was associated with MS susceptibility in Danish cohorts and, then, a replication study was performed with a Norwegian cohort. The latter study also detailed that the association was only observed in the nonprimary-progressive MS forms [29], results validated in further studies [95]. Regarding the HERV-Fc1 expression mechanisms, it has been observed that the transcriptional expression levels of HERV-Fc1 RNA sequences are negatively correlated with the methylation levels of CpG islands on the 5' LTR region and, therefore, a higher HERV-Fc1

An increased expression of HERVs in several autoimmune diseases [21-40, 68] and different types of cancer [14-20], along with the decreased expression levels observed in successfully treated patients with immunomodulatory therapies [88, 89] or chemotherapy [111] point to the potential pathogenic role of HERVs and their putative consideration as a good target for

A humanized monoclonal antibody anti-Env-SU MSRV/HERV-W, GNbAC1, has been studied as a putative MS treatment due to its potential neuroprotector effects [112-115]. The results of a phase IIa clinical trial [114] show that the GNbAC1 treatment blocks the transcription of proinflammatory genes mediated by Env, prevents the formation of nitrosantine, and restores OPC differentiation. Furthermore, GNbAC1 has advantages compared to other MS treatments, because the patients retain all their immune capacity. This treatment has also been studied in

The proteins encoded by HERV-K *env* have been proposed as therapeutic targets for different types of cancer, due to the fact that a general hypomethylation of HERVs sequences has been observed, as well as an increased expression of Np9 and Rec proteins originating from HERV-K in different cancer cells [116]. Both proteins bind to the PLZF protein, a transcriptional repressor of the C-MYC proto-oncogen. The inflammation and the deregulation of protooncogen signaling caused by the HERV-K protein results in a protumorogenic microenviron‐ ment, which favors cell proliferation and metastasis [116]. The use of monoclonal antibodies against HERV-K Env protein inhibits tumoral growth and induces apoptosis in breast cancer cells *in vitro* [116]; therefore, it could be considered a good candidate as a therapy used together

expression involves DNA demethylation [11, 110].

**4. HERVs as future treatment options**

other diseases like diabetes and schizophrenia.

with other cancer treatments.

new treatments.

178 Advances in Molecular Retrovirology

This work aimed to provide a systematic revision of HERVs, with particular emphasis on their potential pathogenic role in MS. Although many aspects of the etiology of this disease remain to be solved, different works support the relevance that HERVs may have in the etiopatho‐ genesis of autoimmune diseases, and specifically in MS. HERVs may contribute to both, disease onset and maintenance, through an exacerbated activation of the immune system. Recently, the results of a phase IIa clinical trial that studies the effectiveness of a human monoclonal antibody (GNbAC1) as a therapeutic target in MS have been published with promising outcome. Thus, evidences support the role of HERVs as potential therapeutic armory in different autoimmune diseases, cancer, and HIV.

#### **Author details**

Belén de la Hera and Elena Urcelay\*

\*Address all correspondence to: elena.urcelay@salud.madrid.org

Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, IdISSC, Madrid, Spain

#### **References**

[1] Ribet D, Heidman T. Formation et évolution des rétrovirus endogènes. *Virologie*. 2010;14:141-50. DOI:10.1684/vir.2010.0294.


nadoblastoma-derived germ cell tumours. *Virchows Arch*. 1999;434:11-5. DOI:10.1007/ s004280050298

[16] Lower R, Lower J, Frank H, Harzmann R, Kurth R. Human teratocarcinomas cul‐ tured in vitro produce unique retrovirus-like viruses. *J Gen Virol*. 1984;65 (Pt 5): 887-98.

[2] Coffin JM. Structure, replication, and recombination of retrovirus genomes: some

[3] Chuong EB. Retroviruses facilitate the rapid evolution of the mammalian placenta.

[4] Perron H, Bernard C, Bertrand JB, Lang AB, Popa I, Sanhadji K, et al. Endogenous retroviral genes, Herpesviruses and gender in Multiple Sclerosis. *J Neurol Sci*.

[5] Young GR, Stoye JP, Kassiotis G. Are human endogenous retroviruses pathogenic? An approach to testing the hypothesis. *Bioessays*. 2013;35:794-803. DOI: 10.1002/bies.

[6] Douville RN, Nath A. Human endogenous retroviruses and the nervous system. *Handb Clin Neurol*. 2014;123:465-85. DOI:10.1016/B978-0-444-53488-0.00022-5.

[7] Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial se‐ quencing and analysis of the human genome. *Nature*. 2001;409:860-921. DOI:

[8] Belshaw R, Katzourakis A, Paces J, Burt A, Tristem M. High copy number in human endogenous retrovirus families is associated with copying mechanisms in addition to

[9] Voisset C, Weiss RA, Griffiths DJ. Human RNA "rumor" viruses: the search for novel human retroviruses in chronic disease. *Microbiol Mol Biol Rev*. 2008;72:157-96. DOI:

[10] Gifford R, Tristem M. The evolution, distribution and diversity of endogenous retro‐

[11] Laska MJ, Nissen KK, Nexo BA. (Some) cellular mechanisms influencing the tran‐ scription of human endogenous retrovirus, HERV-Fc1. *PLoS One*. 2013;8:e53895. DOI:

[12] Patel MR, Emerman M, Malik HS. Paleovirology - ghosts and gifts of viruses past.

[13] Dupressoir A, Heidmann T. [Syncytins – retroviral envelope genes captured for the benefit of placental development]. *Med Sci* (Paris). 2011;27:163-9. DOI:10.1051/meds‐

[14] Buscher K, Trefzer U, Hofmann M, Sterry W, Kurth R, Denner J. Expression of hu‐ man endogenous retrovirus K in melanomas and melanoma cell lines. *Cancer Res*.

[15] Herbst H, Kuhler-Obbarius C, Lauke H, Sauter M, Mueller-Lantzsch N, Harms D, et al. Human endogenous retrovirus (HERV)-K transcripts in gonadoblastomas and go‐

reinfection. *Mol Biol Evol*. 2005;22:814-7. DOI:10.1093/molbev/msi088.

viruses. *Virus Genes*. 2003;26:291-315. DOI:10.1023/A:1024455415443.

*Curr Opin Virol*. 2011;1:304-9. DOI:10.1016/j.coviro.2011.06.007.

2005;65:4172-80. DOI: 10.1158/0008-5472.CAN-04-2983

unifying hypotheses. *J Gen Virol*. 1979;42:1-26.

2009;286:65-72.

180 Advances in Molecular Retrovirology

201300049.

10.1038/35057062.

10.1128/MMBR.00033-07.

10.1371/journal.pone.0053895.

ci/2011272163.

*Bioessays*. 2013;35:853-61. DOI: 10.1002/bies.201300059.


[37] Tai AK, O'Reilly EJ, Alroy KA, Simon KC, Munger KL, Huber BT, et al. Human en‐ dogenous retrovirus-K18 Env as a risk factor in multiple sclerosis. *Mult Scler*. 2008;14:1175-80. DOI:10.1177/1352458508094641.

[27] Dolei A, Perron H. The multiple sclerosis-associated retrovirus and its HERV-W en‐ dogenous family: a biological interface between virology, genetics, and immunology in human physiology and disease. *J Neurovirol*. 2009;15:4-13. DOI:

[28] Dolei A, Serra C, Mameli G, Pugliatti M, Sechi G, Cirotto MC, et al. Multiple sclero‐ sis-associated retrovirus (MSRV) in Sardinian MS patients. *Neurology*. 2002;58:471-3.

[29] Hansen B, Oturai AB, Harbo HF, Celius EG, Nissen KK, Laska MJ, et al. Genetic asso‐ ciation of multiple sclerosis with the marker rs391745 near the endogenous retroviral locus HERV-Fc1: analysis of disease subtypes. *PLoS One*. 2011;6:e26438. DOI:10.1371/

[30] Laska MJ, Brudek T, Nissen KK, Christensen T, Moller-Larsen A, Petersen T, et al. Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis. *J Virol*. 2012;86:3713-22. DOI:10.1128/JVI.06723-11. [31] Mameli G, Astone V, Arru G, Marconi S, Lovato L, Serra C, et al. Brains and periph‐ eral blood mononuclear cells of multiple sclerosis (MS) patients hyperexpress MS-as‐ sociated retrovirus/HERV-W endogenous retrovirus, but not Human herpesvirus 6. *J*

[32] Mameli G, Astone V, Khalili K, Serra C, Sawaya BE, Dolei A. Regulation of the syn‐ cytin-1 promoter in human astrocytes by multiple sclerosis-related cytokines. *Virolo‐*

[33] Rolland A, Jouvin-Marche E, Saresella M, Ferrante P, Cavaretta R, Creange A, et al. Correlation between disease severity and in vitro cytokine production mediated by MSRV (multiple sclerosis associated retroviral element) envelope protein in patients with multiple sclerosis. *J Neuroimmunol*. 2005;160:195-203. DOI:10.1016/j.jneuroim.

[34] Ruprecht K, Obojes K, Wengel V, Gronen F, Kim KS, Perron H, et al. Regulation of human endogenous retrovirus W protein expression by herpes simplex virus type 1: implications for multiple sclerosis. *J Neurovirol*. 2006;12:65-71. DOI:

[35] Schmitt K, Richter C, Backes C, Meese E, Ruprecht K, Mayer J. Comprehensive analy‐ sis of human endogenous retrovirus group HERV-W locus transcription in multiple sclerosis brain lesions by high-throughput amplicon sequencing. *J Virol*.

[36] Sotgiu S, Arru G, Mameli G, Serra C, Pugliatti M, Rosati G, et al. Multiple sclerosisassociated retrovirus in early multiple sclerosis: a six-year follow-up of a Sardinian

cohort. *Mult Scler*. 2006;12:698-703. DOI:10.1177/1352458506070773

*Gen Virol*. 2007;88:264-74. DOI: 10.1099/vir.0.81890-0

*gy*. 2007;362:120-30. DOI:10.1016/j.virol.2006.12.019.

10.1080/13550280802448451.

182 Advances in Molecular Retrovirology

DOI:10.1212/WNL.58.3.471.

journal.pone.0026438.

2004.10.019.

10.1080/13550280600614973.

2013;87:13837-52. DOI:10.1128/JVI.02388-13..


ed with multiple sclerosis is mediated by TLR4. *Int Immunol*. 2015;27. DOI:10.1093/ intimm/dxv025.

[60] Hurst T, Magiorkinis G. Activation of the innate immune response by endogenous retroviruses. *J Gen Virol*. 2014. DOI:10.1099/jgv.0.000017.

[48] Manghera M, Douville RN. Endogenous retrovirus-K promoter: a landing strip for inflammatory transcription factors? *Retrovirology*. 2013;10:16. DOI:

[49] Matouskova M, Blazkova J, Pajer P, Pavlicek A, Hejnar J. CpG methylation suppress‐ es transcriptional activity of human syncytin-1 in non-placental tissues. *Exp Cell Res*.

[50] Reiss D, Zhang Y, Mager DL. Widely variable endogenous retroviral methylation levels in human placenta. *Nucleic Acids Res*. 2007;35:4743-54. DOI: 10.1093/nar/

[51] Hawkes CH, Giovannoni G, Keir G, Cunnington M, Thompson EJ. Seroprevalence of herpes simplex virus type 2 in multiple sclerosis. *Acta Neurol Scand*. 2006;114:363-7.

[52] Frank O, Jones-Brando L, Leib-Mosch C, Yolken R, Seifarth W. Altered transcription‐ al activity of human endogenous retroviruses in neuroepithelial cells after infection

[53] Li F, Nellaker C, Sabunciyan S, Yolken RH, Jones-Brando L, Johansson AS, et al. Transcriptional derepression of the ERVWE1 locus following influenza A virus infec‐

[54] Kwun HJ, Han HJ, Lee WJ, Kim HS, Jang KL. Transactivation of the human endoge‐ nous retrovirus K long terminal repeat by herpes simplex virus type 1 immediate

[55] Lee WJ, Kwun HJ, Kim HS, Jang KL. Activation of the human endogenous retrovirus W long terminal repeat by herpes simplex virus type 1 immediate early protein 1.

[56] Misra A, Chosdol K, Sarkar C, Mahapatra AK, Sinha S. Alteration of a sequence with homology to human endogenous retrovirus (HERV-K) in primary human glioma: implications for viral repeat mediated rearrangement. *Mutat Res*. 2001;484:53-9. DOI:

[57] Kremer D, Schichel T, Forster M, Tzekova N, Bernard C, van der Valk P, et al. Hu‐ man endogenous retrovirus type W envelope protein inhibits oligodendroglial pre‐ cursor cell differentiation. *Ann Neurol*. 2013;74:721-32. DOI: 10.1002/ana.23970..

[58] Rolland A, Jouvin-Marche E, Viret C, Faure M, Perron H, Marche PN. The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses. *J Immunol*. 2006;176:7636-44.

[59] Duperray A, Barbe D, Raguenez G, Weksler BB, Romero IA, Couraud PO, et al. In‐ flammatory response of endothelial cells to a human endogenous retrovirus associat‐

with Toxoplasma gondii. *J Infect Dis*. 2006;194:1447-9. DOI:10.1086/508496.

tion. *J Virol*. 2014;88:4328-37. DOI:10.1128/JVI.03628-13..

10.1186/1742-4690-10-16.

gkm455.

184 Advances in Molecular Retrovirology

2006;312:1011-20. DOI:10.1016/j.yexcr.2005.12.010.

DOI:10.1111/j.1600-0404.2006.00677.x.

early protein 0. *Virus Res*. 2002;86:93-100.

*Mol Cells*. 2003;15:75-80.

10.1016/S0027-5107(01)00240-8.

DOI: 10.4049/jimmunol.176.12.7636


[83] Varadé J, García-Montojo M, de la Hera B, Camacho I, García-Martíneza MA, Arroyo R, et al. Multiple sclerosis retrovirus-like envelope gene: Role of the chromosome 20 insertion. *BBA Clin*. 2015;3:162-7. DOI:10.1016/j.bbacli.2015.02.002.

[72] IMSGC, Hafler DA, Compston A, Sawcer S, Lander ES, Daly MJ, et al. Risk alleles for multiple sclerosis identified by a genomewide study. *N Engl J Med*. 2007;357:851-62.

[73] IMSGC, Sawcer S, Hellenthal G, Pirinen M, Spencer CC, Patsopoulos NA, et al. Ge‐ netic risk and a primary role for cell-mediated immune mechanisms in multiple scle‐

[74] Gourraud PA, Harbo HF, Hauser SL, Baranzini SE. The genetics of multiple sclerosis: an up-to-date review. *Immunol Rev*. 2012;248:87-103. DOI: 10.1111/j.1600-065X.

[75] IMSGC, Beecham AH, Patsopoulos NA, Xifara DK, Davis MF, Kemppinen A, et al. Analysis of immune-related loci identifies 48 new susceptibility variants for multiple

[76] Perron H, Geny C, Laurent A, Mouriquand C, Pellat J, Perret J, et al. Leptomeningeal cell line from multiple sclerosis with reverse transcriptase activity and viral particles.

[77] Perron H, Garson JA, Bedin F, Beseme F, Paranhos-Baccala G, Komurian-Pradel F, et al. Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis. *Proc*

[78] Pavlicek A, Paces J, Elleder D, Hejnar J. Processed pseudogenes of human endoge‐ nous retroviruses generated by LINEs: their integration, stability, and distribution.

[79] Mameli G, Poddighe L, Astone V, Delogu G, Arru G, Sotgiu S, et al. Novel reliable real-time PCR for differential detection of MSRVenv and syncytin-1 in RNA and DNA from patients with multiple sclerosis. *J Virol Methods*. 2009;161:98-106. DOI:

[80] Roebke C, Wahl S, Laufer G, Stadelmann C, Sauter M, Mueller-Lantzsch N, et al. An N-terminally truncated envelope protein encoded by a human endogenous retrovi‐ rus W locus on chromosome Xq22.3. *Retrovirology*. 2010;7:69. DOI:

[81] Laufer G, Mayer J, Mueller BF, Mueller-Lantzsch N, Ruprecht K. Analysis of transcri‐ bed human endogenous retrovirus W env loci clarifies the origin of multiple sclero‐ sis-associated retrovirus env sequences. *Retrovirology*. 2009;6:37. DOI:

[82] Garcia-Montojo M, de la Hera B, Varade J, de la Encarnacion A, Camacho I, Domi‐ nguez-Mozo M, et al. HERV-W polymorphism in chromosome X is associated with multiple sclerosis risk and with differential expression of MSRV. *Retrovirology*.

rosis. *Nature*. 2011;476:214-9. DOI:10.1038/nature10251.

sclerosis. *Nat Genet*. 2013;45:1353-60. DOI:10.1038/ng.2770..

2012.01134.x.

186 Advances in Molecular Retrovirology

*Res Virol*. 1989;140:551-61.

*Natl Acad Sci U S A*. 1997;94:7583-8.

10.1016/j.jviromet.2009.05.024..

10.1186/1742-4690-7-69.

10.1186/1742-4690-6-37.

2014;11:2.

*Genome Res*. 2002;12:391-9. DOI:10.1101/gr.216902


susceptibility: study in the Spanish population and meta-analysis. *PLoS One*. 2013;8:e62090. DOI:10.1371/journal.pone.0062090..


[105] Nielsen TR, Pedersen M, Rostgaard K, Frisch M, Hjalgrim H. Correlations between Epstein-Barr virus antibody levels and risk factors for multiple sclerosis in healthy individuals. *Mult Scler*. 2007;13:420-3. DOI:10.1177/1352458506071470

susceptibility: study in the Spanish population and meta-analysis. *PLoS One*.

[94] Nexo BA, Christensen T, Frederiksen J, Moller-Larsen A, Oturai AB, Villesen P, et al. The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1. *PLoS One*. 2011;6:e16652. DOI: 10.1371/jour‐

[95] de la Hera B, Varade J, Garcia-Montojo M, Alcina A, Fedetz M, Alloza I, et al. Human endogenous retrovirus HERV-Fc1 association with multiple sclerosis susceptibility: a

[96] Kraus B, Fischer K, Sliva K, Schnierle BS. Vaccination directed against the human en‐ dogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system. *Virol J*. 2014;11:58. DOI:10.1186/1743-422X-11-58.

[97] Turcanova VL, Bundgaard B, Hollsberg P. Human herpesvirus-6B induces expres‐ sion of the human endogenous retrovirus K18-encoded superantigen. *J Clin Virol*.

[98] Tai AK, Luka J, Ablashi D, Huber BT. HHV-6A infection induces expression of HERV-K18-encoded superantigen. *J Clin Virol*. 2009;46:47-8. DOI:10.1016/j.jcv.

[99] Hsiao FC, Lin M, Tai A, Chen G, Huber BT. Cutting edge: Epstein-Barr virus transac‐ tivates the HERV-K18 superantigen by docking to the human complement receptor 2 (CD21) on primary B cells. *J Immunol*. 2006;177:2056-60. DOI: 10.4049/ jimmunol.

[100] Sutkowski N, Conrad B, Thorley-Lawson DA, Huber BT. Epstein-Barr virus transac‐ tivates the human endogenous retrovirus HERV-K18 that encodes a superantigen.

[101] Hsiao FC, Tai AK, Deglon A, Sutkowski N, Longnecker R, Huber BT. EBV LMP-2A employs a novel mechanism to transactivate the HERV-K18 superantigen through its

[102] Sutkowski N, Chen G, Calderon G, Huber BT. Epstein-Barr virus latent membrane protein LMP-2A is sufficient for transactivation of the human endogenous retrovirus HERV-K18 superantigen. *J Virol*. 2004;78:7852-60. DOI:10.1128/JVI.

[103] Buljevac D, van Doornum GJ, Flach HZ, Groen J, Osterhaus AD, Hop W, et al. Ep‐ stein-Barr virus and disease activity in multiple sclerosis. *J Neurol Neurosurg Psychia‐*

[104] Christensen T. The role of EBV in MS pathogenesis. *Int MS J*. 2006;13:52-7.

*Immunity*. 2001;15:579-89. DOI:10.1016/S1074-7613(01)00210-2.

ITAM. *Virology*. 2009;385:261-6. DOI:10.1016/j.virol.2008.11.025.

*try*. 2005;76:1377-81. DOI:10.1136/jnnp.2004.048504

meta-analysis. *PLoS One*. 2014;9:e90182. DOI:10.1371/journal.pone.0090182.

2013;8:e62090. DOI:10.1371/journal.pone.0062090..

2009;46:15-9. DOI:10.1016/j.jcv.2009.05.015.

nal.pone.0016652.

188 Advances in Molecular Retrovirology

2009.05.019.

177.4.2056

78.14.7852-7860.2004


associated endogenous retrovirus: a first-in-humans randomized clinical study. *Clin Ther*. 2012;34:2268-78. DOI:10.1016/j.clinthera.2012.11.006.


associated endogenous retrovirus: a first-in-humans randomized clinical study. *Clin*

[116] Downey RF, Sullivan FJ, Wang-Johanning F, Ambs S, Giles FJ, Glynn SA. Human en‐ dogenous retrovirus K and cancer: Innocent bystander or tumorigenic accomplice?

[117] van der Kuyl AC. HIV infection and HERV expression: a review. *Retrovirology*.

[118] Bhardwaj N, Maldarelli F, Mellors J, Coffin JM. HIV-1 infection leads to increased transcription of human endogenous retrovirus HERV-K (HML-2) proviruses in vivo but not to increased virion production. *J Virol*. 2014;88:11108-20. DOI:10.1128/JVI.

[119] Michaud HA, SenGupta D, de Mulder M, Deeks SG, Martin JN, Kobie JJ, et al. Cut‐ ting edge: An antibody recognizing ancestral endogenous virus glycoproteins medi‐ ates antibody-dependent cellular cytotoxicity on HIV-1-infected cells. *J Immunol*.

*Ther*. 2012;34:2268-78. DOI:10.1016/j.clinthera.2012.11.006.

*Int J Cancer*. 2014;137:1249-57. DOI: 10.1002/ijc.29003.

2014;193:1544-8. DOI:10.4049/jimmunol.1302108.

2012;9:6. DOI:10.1186/1742-4690-9-6.

01623-14.

190 Advances in Molecular Retrovirology

## *Edited by Shailendra K. Saxena*

This book gives a comprehensive overview of recent advances in Retrovirology, as well as general concepts of molecular biology of retroviral infections, immunopathology, diagnosis, and prevention, to current clinical recommendations in management of retroviruses, including endogenous retroviruses, highlighting the ongoing issues, recent advances, with future directions in diagnostic approaches and therapeutic strategies.

Advances in Molecular Retrovirology

Advances in Molecular

Retrovirology

*Edited by Shailendra K. Saxena*

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