**4. Vaccination strategies against HIV-1**

Many infection-related hurdles complicate the development of an HIV vaccine. These include the high genetic variability, the potential of cell-to-cell transmission and other evasion strategies such as down-regulating MHC I in infected cells and latency of the virus [77]. In addition, correlates conferring protection against HIV remain to be established. A number of potential markers have been suggested to prevent or control HIV infection. These comprise: production of high titers of neutralizing antibodies with broad specificities, concomitant HIV-specific activation of CD4+ and CD8+ T cells, polyfunctional T cell responses (production of several immune mediators by the same T cell) and induction of long- term memory cells [78].

#### **4.1. Prophylactic vaccines**

Prophylactic vaccines rely on the production of antibodies that bind to free virus particles thereby preventing viral entry into host cells (defined as neutralization) and thus block infection. Vaccines are designed to mimic natural infections, by using live-attenuated virus (measles, mumps), chemically inactivated virus (polio) or recombinant subunits of the virus (Hepatitis B). There is circumstantial evidence that neutralizing antibodies could play a role in the protection against HIV. HIV-neutralizing IgA antibodies have been isolated in frequently exposed individuals, who remained uninfected [62, 79]. In addition, passive immunization with several HIV neutralizing IgG monoclonal antibodies protects macaques against infectious SHIV (simian immune deficiency virus with an HIV envelope) [80, 81]. Although attenuated SIV vaccines provided some level of protection against super-infection in macaques, attenuated HIV is considered too risky to be ever tried in humans [82]. Therefore, much effort has been invested in the development of subunit vaccines that could elicit production of neutralizing antibodies. It should be taken into account that broadly neutralizing antibodies (bNAbs) that inhibit also heterologous viruses *in vitro*, can be detected in 20% of naturally infected individuals. This implies that the production of these

antibodies is not sufficient to provide full protection against established HIV, but could still be efficient in prevention of cell-free transmission. The most important reason why it is difficult to induce bNAbs is the extreme variability of HIV Env antigenic epitopes. Moreover, the virus is shielded by non-immunogenic glycans, which hinder binding of antibodies to the envelope proteins [83, 84]. Due to these problems, prophylactic vaccine trials in humans have failed to elicit protection. The only exception till now is the recent Thai vaccine trial (RV144).

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 37

Adenoviruses, poxviruses and lentiviruses are the most frequently used viral vector systems. The major advantages of these vectors are the high transduction efficiency resulting in high level expression of the encoded protein and the possibility to target specific cells achieved by altering the viral tropism (e.g. by pseudotyping with envelope or counter receptors of another virus) [113, 114]. Major drawbacks are the high risk of insertional mutagenesis, high production cost of large amounts high-titered viral stocks and a limited size of nucleic acids that can be packed [113, 114]. The first trial of a preventive HIV vaccine that was designed to elicit a strong cellular immune response was the STEP trial done by Merck. It involved immunization of almost 3000 healthy uninfected volunteers with three recombinant adenovirus serotype-5 (rAD5) vectors, Ad5-*gag,* Ad5-*pol* and Ad5-*nef*. Unfortunately, it failed to induce protection against infection [115, 116]. Moreover, the vaccine increased the rate of HIV infection in individuals with pre-existing immunity to adenoviruses [117]. The exact mechanism that underlies this phenomenon remains to be elucidated, but it has been suggested that the activation of pre-existing vector specific T cells may have increased numbers of HIV target cells. This might be avoided by the use of less prevalent adenovirus serotypes (e.g. Ad11, Ad24 or Ad35) instead of the Ad5 vector in

Alternatively, poxvirus-based vectors should be taken into account since they do not pose any problems with pre-existing immunity. In addition, they are used as highly attenuated vaccinia virus strains. Three of the best characterized highly attenuated pox vectors are the recombinant viral canary pox vectors such as the highly attenuated vaccinia virus strain ALVAC [120], the recombinant modified vaccinia Ankara (MVA) vectors [121-123] and canarypox-derived NYVAC [120, 124, 125]. Recombinant pox vectors, encoding HIV antigens, have been shown to be safe in humans and to induce HIV specific immune responses. No protection against HIV infection has been achieved with the exception of the preventive RV144 phase III clinical trial. In this clinical trial, involving 16 000 uninfected individuals, a canary pox vector coding HIV Gag and Env was used as prime immunization followed by a recombinant Env gp120 protein boost (RV144). A 31% efficacy of protection against HIV infection was demonstrated after three years [126]. Very recently, Barouch *et al.* reported that rhesus monkeys, receiving heterologous vector regimes (adeno with MVA),

Another type of vectors that could avoid the pre-existing immunity issue is based on lentiviruses. These vectors have been explored extensively in the field of gene therapy since they efficiently transduce non-dividing cells, such as DCs [128, 129], and promote long term antigen expression [130]. Lentiviral vector vaccines have been shown to induce both high short term and long-term anti-HIV immune responses in mice [131, 132]. Even in the absence of circulating CD4+ T cells, induction of specific CTLs was obtained [133]. Despite reassuring safety and tolerability results in a phase I clinical trial [134], the major concern remains the risk of insertional mutagenesis [135]. Attempts to overcome this risk, have led to the design of self-inactivating vectors, vectors with targeted integration and non-integrating

designing future HIV vaccines [118, 119].

vectors [135].

were protected with an efficacy of 80% after SIV challenges [127].

Nevertheless neutralizing antibodies with activity against easy-to-neutralize so-called "Tier 1" viruses have been induced in a number of animal trials, but these antibodies failed to broaden and faded rapidly, even upon repeated heterologous boosts [85]. The failure to induce high titers of NAbs moved the field towards strategies aiming at stimulating polyfunctional and sustained CD4+ T help responses [69] to support high quality cytotoxic T cells (both central memory and effector memory). These cells would be necessary to rapidly eliminate infected cells, if antibodies fail to prevent cellular infection [86, 87]. This "second line prevention" hypothesis was further supported by the observations that HIV-specific CD4+ and/or CD8+ T cells as well as particular human leukocyte antigen (HLA) class I markers, and not antibodies, correlate with resistance to HIV in some highly exposed seronegative children (potential vertical transmission) [88] or women (potential heterosexual transmission) [89-91].

In this connection, current HIV vaccines are also aiming at the induction (prophylactic field) or enhancing (therapeutic field) of HIV specific T cell responses. Such vaccines would elicit or boost HIV specific cytotoxic T cells (CTLs) to eliminate infected cells and CD4+ T cells, which can help to induce and maintain B cell and CD8+ T cells responses [92]. Several strategies are currently under investigation to establish effective T cell responses in either a preventive or therapeutic setting either based on protein [93, 94] or peptide [95] vaccinations, virus like particles (VPLs) [96], DNA vaccination using viral vectors [97, 98], prime-boost vaccinations [99, 100] or DC-based vaccines [101-109].

#### **4.2. Viral vaccine delivery**

Whereas the use of live attenuated HIV is considered to be unsafe for the use in humans, the development of vaccines based on HIV-inactivated with formalin is compromised by the fact that the antigenicity of the envelope gets lost. Milder formalin treatment of the virus, followed by heat-inactivation has been shown to circumvent this hurdle and induce modest neutralizing antibodies titer in non-human primates [110].

During the last decade, a variety of vaccines was designed using (plasmid) DNA/RNA vaccine candidates for priming followed by live vectored recombinant vaccines for boosting, some of which have already been tested in advanced stages of clinical trials [111, 112]. We will highlight here some of the specific characteristics of viral vectors, which have been used in preclinical and early clinical preventive vaccinations against SIV and HIV, respectively.

Adenoviruses, poxviruses and lentiviruses are the most frequently used viral vector systems. The major advantages of these vectors are the high transduction efficiency resulting in high level expression of the encoded protein and the possibility to target specific cells achieved by altering the viral tropism (e.g. by pseudotyping with envelope or counter receptors of another virus) [113, 114]. Major drawbacks are the high risk of insertional mutagenesis, high production cost of large amounts high-titered viral stocks and a limited size of nucleic acids that can be packed [113, 114]. The first trial of a preventive HIV vaccine that was designed to elicit a strong cellular immune response was the STEP trial done by Merck. It involved immunization of almost 3000 healthy uninfected volunteers with three recombinant adenovirus serotype-5 (rAD5) vectors, Ad5-*gag,* Ad5-*pol* and Ad5-*nef*. Unfortunately, it failed to induce protection against infection [115, 116]. Moreover, the vaccine increased the rate of HIV infection in individuals with pre-existing immunity to adenoviruses [117]. The exact mechanism that underlies this phenomenon remains to be elucidated, but it has been suggested that the activation of pre-existing vector specific T cells may have increased numbers of HIV target cells. This might be avoided by the use of less prevalent adenovirus serotypes (e.g. Ad11, Ad24 or Ad35) instead of the Ad5 vector in designing future HIV vaccines [118, 119].

36 Immunodeficiency

Thai vaccine trial (RV144).

transmission) [89-91].

**4.2. Viral vaccine delivery** 

HIV, respectively.

antibodies is not sufficient to provide full protection against established HIV, but could still be efficient in prevention of cell-free transmission. The most important reason why it is difficult to induce bNAbs is the extreme variability of HIV Env antigenic epitopes. Moreover, the virus is shielded by non-immunogenic glycans, which hinder binding of antibodies to the envelope proteins [83, 84]. Due to these problems, prophylactic vaccine trials in humans have failed to elicit protection. The only exception till now is the recent

Nevertheless neutralizing antibodies with activity against easy-to-neutralize so-called "Tier 1" viruses have been induced in a number of animal trials, but these antibodies failed to broaden and faded rapidly, even upon repeated heterologous boosts [85]. The failure to induce high titers of NAbs moved the field towards strategies aiming at stimulating polyfunctional and sustained CD4+ T help responses [69] to support high quality cytotoxic T cells (both central memory and effector memory). These cells would be necessary to rapidly eliminate infected cells, if antibodies fail to prevent cellular infection [86, 87]. This "second line prevention" hypothesis was further supported by the observations that HIV-specific CD4+ and/or CD8+ T cells as well as particular human leukocyte antigen (HLA) class I markers, and not antibodies, correlate with resistance to HIV in some highly exposed seronegative children (potential vertical transmission) [88] or women (potential heterosexual

In this connection, current HIV vaccines are also aiming at the induction (prophylactic field) or enhancing (therapeutic field) of HIV specific T cell responses. Such vaccines would elicit or boost HIV specific cytotoxic T cells (CTLs) to eliminate infected cells and CD4+ T cells, which can help to induce and maintain B cell and CD8+ T cells responses [92]. Several strategies are currently under investigation to establish effective T cell responses in either a preventive or therapeutic setting either based on protein [93, 94] or peptide [95] vaccinations, virus like particles (VPLs) [96], DNA vaccination using viral vectors [97, 98],

Whereas the use of live attenuated HIV is considered to be unsafe for the use in humans, the development of vaccines based on HIV-inactivated with formalin is compromised by the fact that the antigenicity of the envelope gets lost. Milder formalin treatment of the virus, followed by heat-inactivation has been shown to circumvent this hurdle and induce modest

During the last decade, a variety of vaccines was designed using (plasmid) DNA/RNA vaccine candidates for priming followed by live vectored recombinant vaccines for boosting, some of which have already been tested in advanced stages of clinical trials [111, 112]. We will highlight here some of the specific characteristics of viral vectors, which have been used in preclinical and early clinical preventive vaccinations against SIV and

prime-boost vaccinations [99, 100] or DC-based vaccines [101-109].

neutralizing antibodies titer in non-human primates [110].

Alternatively, poxvirus-based vectors should be taken into account since they do not pose any problems with pre-existing immunity. In addition, they are used as highly attenuated vaccinia virus strains. Three of the best characterized highly attenuated pox vectors are the recombinant viral canary pox vectors such as the highly attenuated vaccinia virus strain ALVAC [120], the recombinant modified vaccinia Ankara (MVA) vectors [121-123] and canarypox-derived NYVAC [120, 124, 125]. Recombinant pox vectors, encoding HIV antigens, have been shown to be safe in humans and to induce HIV specific immune responses. No protection against HIV infection has been achieved with the exception of the preventive RV144 phase III clinical trial. In this clinical trial, involving 16 000 uninfected individuals, a canary pox vector coding HIV Gag and Env was used as prime immunization followed by a recombinant Env gp120 protein boost (RV144). A 31% efficacy of protection against HIV infection was demonstrated after three years [126]. Very recently, Barouch *et al.* reported that rhesus monkeys, receiving heterologous vector regimes (adeno with MVA), were protected with an efficacy of 80% after SIV challenges [127].

Another type of vectors that could avoid the pre-existing immunity issue is based on lentiviruses. These vectors have been explored extensively in the field of gene therapy since they efficiently transduce non-dividing cells, such as DCs [128, 129], and promote long term antigen expression [130]. Lentiviral vector vaccines have been shown to induce both high short term and long-term anti-HIV immune responses in mice [131, 132]. Even in the absence of circulating CD4+ T cells, induction of specific CTLs was obtained [133]. Despite reassuring safety and tolerability results in a phase I clinical trial [134], the major concern remains the risk of insertional mutagenesis [135]. Attempts to overcome this risk, have led to the design of self-inactivating vectors, vectors with targeted integration and non-integrating vectors [135].

Replicating and persistent recombinant cytomegalovirus (CMV) vectors have recently been shown to be a promising system in rhesus macaques [136]. Prophylactically vaccinated animals maintained CD4+ and CD8+ T effector memory (TEM) cell responses, regardless to pre-existing CMV immunity, and were more resistant to challenge than the control group even in the absence of neutralizing antibodies [137]. The authors suggest that TEM responses are crucial in the protection against HIV infection after sexual exposure. This is obviously also the scope of HIV immunotherapy where sustained effector and memory T cells can eliminate infected cells.

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 39

immunotherapy [148], which constituted a model for development of similar HIV

DCs are the sentinels of the immune system, bridging innate and adaptive immunity, in response to pathogens crossing the mucosal or dermal barrier. Immature DCs (iDCs) continuously sample their environment and take up autologous and foreign antigens [149]. They undergo maturation in response to signals that originate from pathogen-associated molecular patterns (PAMPs). These PAMPs activate a set of pattern recognition receptors (PRRs) such as Toll like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-lectin receptors (CLRs) and retinoic acid-inducible gene protein (RIG)- like receptors (RLRs). Triggering of PPR results in an increased expression of major histocompatibility complex I and II (MHC I and MHC II), co-stimulatory molecules (CD80 and CD86) as well as secretion of T cell stimulatory cytokines (e.g. IL-12). During this maturation process DCs lose their ability to take up antigens and chemokine receptors (e.g. CCR7) are up-regulated in order to promote their migration to lymph nodes. Mature DCs process endogenous antigens via proteasome into 8-9 amino-acid peptides which are then loaded on MHC I and presented to CD8+ T cells. Exogenous antigens are processed via the endolysosome into longer peptides to be load onto MHC II for presentation to CD4+ T cells. The capacity of DCs to present exogenous antigens also via MHC I pathway (i.e. cross presentation), distinguishes them from other APCs, such as macrophages and B cells. To stimulate effective T cell responses, peptide-MHC complex on DCs should interact with T cell receptors (TCR). This is accompanied by binding of co-stimulatory molecules on DCs with CD28 present on T cells (**figure 3**). Finally, produced cytokines determine the differentiation of the effector cells into Th1, Th2 or CTL [150]. The latter is achieved after DCs' licensing by the interaction of CD40 on the mature DCs with CD40L expressed on CD4+ T cells. IL-4 secretion promotes CD4+ Th2 cells, stimulating the production of antibody producing B cells. IL-12 promotes CD4+ Th1 cells, providing help to CTL to kill infected cells. Secretion of IL-10 has a negative impact on Th1 or Th2 cells and induces immune tolerance. Licensed DCs also induce differentiation of CD8+ T cells into CTL via peptide-MHC I complex and promote survival of

CD8+ T cells via co-stimulation through CD137L (4-1BBL) [151].

Roughly five DC subsets can be distinguished [152]. Classical or tissue resident DCs are located in lymphoid organs such as spleen and lymph nodes. Migratory DCs, found in nonlymphoid organs such as skin, intestines and lungs, sample their environment and migrate to lymph nodes to present tissue derived antigen to T cells. Langerhans cells reside in the multi-layerd epithelium of the skin, oral and genital surfaces. Plasmacytoid DCs (pDCs) and myeloid or monocyte-derived DCs may be present in various tissues, yet they mainly circulate in the blood. pDCs are known as major producers of type I interferons (IFNs) in response to virus-associated molecules such as single-stranded (ss) RNA and unmethylated cytosine-phosphate-guanine (CpG)-rich DNA that trigger TLR7 and TLR9, respectively [153]. Myeloid DCs represent the major fraction of APCs in the blood that responds to TLR ligation by producing IL-12 [154]. Noteworthy, the APC function of DCs is impaired by

immunotherapies.

**5.2. Dendritic cells** 

Virus-like-particles (VLPs) have recently emerged as novel delivery systems. They contain envelope and core proteins from SIV/HIV in their native structure. These pseudo-virions are produced in baculovirus or vaccinia virus expression systems where Gag and Env proteins from HIV or SIV are co-expressed and spontaneously assembled. The immunogenicity of these vaccines was only modest in non-human primates [96], however, efficiency was greatly improved when combined with a HIV DNA vaccine prime [138].

Safety concerns and difficulties related to repeated administrations of viral vectors that may evoke dangerous immune reactions are the most important bottlenecks in regard to clinical application in humans. To improve the general safety profile and circumventing the drawbacks inherited to viral delivery, well-defined particulate vaccines have emerged as promising candidates in the field of vaccine development.
