**5. HIV Immunotherapy with DC-based vaccines**

#### **5.1. Therapeutic vaccines**

Since the introduction of HAART, HIV-1 infection has evolved into a chronic but treatable disease. Although HAART suppresses viral replication and partially reconstitutes both CD4+ T cell numbers [139] and T cell immune responses to opportunistic infections, it cannot restore effective HIV specific T cell responses [140], resulting in a rapid rebound of HIV-1 replication upon treatment interruption [141]. This implies that infected individuals are bound to lifelong treatment, imposing a high burden in terms of adherence, costs and the risk of drug related metabolic disorders [142]. Therefore, therapeutic vaccination has emerged as an option to boost and improve the cellular immune responses in infected individuals [143]. This concept is supported by increasing evidence that strong HIV-1 specific CD4+ helper T cells and CD8+ CTLs are responsible for viral control both in macaques [144] and humans [145]. During acute infection CD8+ T cell responses contribute to control of the initial viraemia peak. However, in most infected subjects, this CD8+ T cell response loses its efficacy during chronic infection. This is due to a high mutation rate of the virus, the absence of proliferative and highly functional CD4+ T cells [145, 146] and the appearance of impaired or apoptotic HIV-specific CD8 T cells [147]. Clearly, the major challenge for therapeutic vaccination is to elicit strong CD4+ and CD8+ responses that would allow stopping of HAART. I*n vitro* modulation of DCs to efficiently present target antigens to elicit cellular immune responses has been shown to be a promising strategy in cancer immunotherapy [148], which constituted a model for development of similar HIV immunotherapies.

#### **5.2. Dendritic cells**

38 Immunodeficiency

eliminate infected cells.

**5.1. Therapeutic vaccines** 

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

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

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

Since the introduction of HAART, HIV-1 infection has evolved into a chronic but treatable disease. Although HAART suppresses viral replication and partially reconstitutes both CD4+ T cell numbers [139] and T cell immune responses to opportunistic infections, it cannot restore effective HIV specific T cell responses [140], resulting in a rapid rebound of HIV-1 replication upon treatment interruption [141]. This implies that infected individuals are bound to lifelong treatment, imposing a high burden in terms of adherence, costs and the risk of drug related metabolic disorders [142]. Therefore, therapeutic vaccination has emerged as an option to boost and improve the cellular immune responses in infected individuals [143]. This concept is supported by increasing evidence that strong HIV-1 specific CD4+ helper T cells and CD8+ CTLs are responsible for viral control both in macaques [144] and humans [145]. During acute infection CD8+ T cell responses contribute to control of the initial viraemia peak. However, in most infected subjects, this CD8+ T cell response loses its efficacy during chronic infection. This is due to a high mutation rate of the virus, the absence of proliferative and highly functional CD4+ T cells [145, 146] and the appearance of impaired or apoptotic HIV-specific CD8 T cells [147]. Clearly, the major challenge for therapeutic vaccination is to elicit strong CD4+ and CD8+ responses that would allow stopping of HAART. I*n vitro* modulation of DCs to efficiently present target antigens to elicit cellular immune responses has been shown to be a promising strategy in cancer

greatly improved when combined with a HIV DNA vaccine prime [138].

promising candidates in the field of vaccine development.

**5. HIV Immunotherapy with DC-based vaccines** 

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 HIV, which could contribute to the dysfunction of HIV specific T cell responses since maturation of DCs and IL-12 secretion are diminished, thereby suppressing T cell responses [155]. HIV-infected DCs preferably secrete IL-10 thus limiting T cell proliferation and activation and rather induce tolerance [156]. HIV-1 infected DCs also act as a Trojan horse to infect new T cells by promoting tolerance of T cells to HIV-1 [157].

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 41

Suppression of viral load was correlated with HIV specific IL-2 or IFN-γ producing CD4+ T cells and perforin producing CD8+

Lengthening of viral rebound correlated with numbers of HIVspecific proliferative CD4+ and

Inverse correlation between decrease in viral load and HIVspecific T cell responses. [103]

In 3/6 cases Env-specific and proliferative immune responses

In 2/4 patients moderate CD8+ T cell responses were observed. No

lower viral setpoints after therapy interruption. [105]

were observed. [107]

effector T cells. [108]

CD8+ T cells. [104]

T cell responses in the absence of virological responses was shown [101, 105], whereas in the third study a transient decrease in viral load was found in 50% of vaccinated individuals [106]. Lu *et al.* were the first to demonstrate *in vivo* that a vaccine made of autologous monocyte-derived DCs, pulsed with autologous, aldithriol-2-inactivated HIV-1 was capable of inducing HIV-1-specific T cell responses. Moreover, there was an 80% decrease in plasma viral load levels over the first 112 days after vaccination. A prolonged suppression of viral load (of more than 90%) was seen for at least 1 year after vaccination in 8 out of 19 vaccinated patients. This suppression of viral load correlated with HIV-1-specific IL-2 and IFN-γ expressing CD4+ T cells and with HIV-1 Gag-specific perforin-expressing CD8+ effector cells [108]. Unfortunately, the lack of a control group does not allow a proper evaluation of the results obtained. The group of García *et al.* performed two clinical trials with autologous DCs pulsed with heat-inactivated virus [103, 104]. In a double blind placebo controlled study with a similar setup as Lu *et al*., García *et al.* observed a small but significant decrease in viral loads for at least 48 weeks, concomitant with a weak increase in HIV-1 specific T cell responses in therapy naïve HIV infected individuals [103]. Another study performed by the same group in HAART treated patients showed a lengthening of the rebound after therapy interruption and a moderate increase in HIV specific T cell

**Vaccine strategy Subjects Clinical trial outcome** 

HAART treated patients followed by therapy interruption (n=18)

Untreated patients (n= 18)

Untreated patients (n= 6)

HAART treated patients followed by therapy interruption (n=4)

Untreated patients (n= 18)

responses [104].

**Form of antigen** 

**Inactivated virus** 

DCs pulsed with autologous AT2 inactivated virus (109)

DCs pulsed with autologous heat inactivated virus (106)

DCs pulsed with autologous heat inactivated virus (109)

Pol and Env peptides

DCs pulsed with HLA A\*0201 binding epitopes in Gag, Nef and Env

**Peptides** DCs pulsed with Gag,

**Figure 3.** Differentiation process of dendritic cells. Immature DCs take up antigens. Activation of PPRs results in the differentiation towards mature DCs. The co-stimulatory molecules (B7-1, and B7-2) are overexpressed and the expression of the lymph node homing receptor CCR7 is induced. Mature DCs migrate to the draining lymph node, where they present antigens to cognate CD4+ T cells. Cross-linking CD40 on the DCs by CD40L expressed on the antigen-activated CD4+ T cell, induces the mature DCs to differentiate further, a process known as licensing. Licensed DCs up-regulate additional cell surface proteins, such as CD137L. The licensed DCs now present antigens to cognate CD8+ T cells. CD137L– mediated co-stimulation through CD137 on the antigen-activated CD8+ T cells enhances the survival and proliferative capacity of the activated CD8+ T cells.

#### **5.3. HIV-1 antigen loaded dendritic cells tested in clinical trails**

To obtain a large population of DCs to be used in immunotherapy, DCs are derived from blood monocytes that are cultured in the presence of granulocyte and macrophage colony stimulation factor (GM-CSF) and IL-4 [158, 159]. Various approaches for loading DCs with antigens have been applied. They include the use of inactivated virus [104, 160], recombinant viral proteins [107, 161-163], peptides [101, 164], DNA [165-167] and mRNA [168-172]. After loading, DCs are matured using various maturation cocktails composed of cytokines (such as type 1 (α,β) or type 2 (γ) interferons, TNF-α, IL-6) prostaglandines (PG), TLR ligands, T cell derived products (CD40L) or small interfering RNA (siRNA) against suppressors of cytokine signaling (SOCS)-1 [98, 151].

To this date at least ten DC-based immunotherapeutic vaccine trials (**table 2**) have been tested in infected individuals (recently reviewed by García and Routy [173]. Kundu *et al*. were the first to perform a human clinical trial, in which treatment-naïve HIV infected individuals were vaccinated with protein or peptides pulsed autologous DCs [107]. The vaccine was well tolerated and resulted in an increase of HIV specific CD8+ T cell response in 3 out of 6 individuals, but no effect on viral load was observed. Three other clinical trials tested peptides pulsed DCs [101, 105, 106]. In two of these trials an increase in HIV-specific T cell responses in the absence of virological responses was shown [101, 105], whereas in the third study a transient decrease in viral load was found in 50% of vaccinated individuals [106]. Lu *et al.* were the first to demonstrate *in vivo* that a vaccine made of autologous monocyte-derived DCs, pulsed with autologous, aldithriol-2-inactivated HIV-1 was capable of inducing HIV-1-specific T cell responses. Moreover, there was an 80% decrease in plasma viral load levels over the first 112 days after vaccination. A prolonged suppression of viral load (of more than 90%) was seen for at least 1 year after vaccination in 8 out of 19 vaccinated patients. This suppression of viral load correlated with HIV-1-specific IL-2 and IFN-γ expressing CD4+ T cells and with HIV-1 Gag-specific perforin-expressing CD8+ effector cells [108]. Unfortunately, the lack of a control group does not allow a proper evaluation of the results obtained. The group of García *et al.* performed two clinical trials with autologous DCs pulsed with heat-inactivated virus [103, 104]. In a double blind placebo controlled study with a similar setup as Lu *et al*., García *et al.* observed a small but significant decrease in viral loads for at least 48 weeks, concomitant with a weak increase in HIV-1 specific T cell responses in therapy naïve HIV infected individuals [103]. Another study performed by the same group in HAART treated patients showed a lengthening of the rebound after therapy interruption and a moderate increase in HIV specific T cell responses [104].

40 Immunodeficiency

HIV, which could contribute to the dysfunction of HIV specific T cell responses since maturation of DCs and IL-12 secretion are diminished, thereby suppressing T cell responses [155]. HIV-infected DCs preferably secrete IL-10 thus limiting T cell proliferation and activation and rather induce tolerance [156]. HIV-1 infected DCs also act as a Trojan horse to

**Figure 3.** Differentiation process of dendritic cells. Immature DCs take up antigens. Activation of PPRs results in the differentiation towards mature DCs. The co-stimulatory molecules (B7-1, and B7-2) are overexpressed and the expression of the lymph node homing receptor CCR7 is induced. Mature DCs migrate to the draining lymph node, where they present antigens to cognate CD4+ T cells. Cross-linking CD40 on the DCs by CD40L expressed on the antigen-activated CD4+ T cell, induces the mature DCs to differentiate further, a process known as licensing. Licensed DCs up-regulate additional cell surface proteins, such as CD137L. The licensed DCs now present antigens to cognate CD8+ T cells. CD137L– mediated co-stimulation through CD137 on the antigen-activated CD8+ T cells enhances the survival and

To obtain a large population of DCs to be used in immunotherapy, DCs are derived from blood monocytes that are cultured in the presence of granulocyte and macrophage colony stimulation factor (GM-CSF) and IL-4 [158, 159]. Various approaches for loading DCs with antigens have been applied. They include the use of inactivated virus [104, 160], recombinant viral proteins [107, 161-163], peptides [101, 164], DNA [165-167] and mRNA [168-172]. After loading, DCs are matured using various maturation cocktails composed of cytokines (such as type 1 (α,β) or type 2 (γ) interferons, TNF-α, IL-6) prostaglandines (PG), TLR ligands, T cell derived products (CD40L) or small interfering RNA (siRNA) against

To this date at least ten DC-based immunotherapeutic vaccine trials (**table 2**) have been tested in infected individuals (recently reviewed by García and Routy [173]. Kundu *et al*. were the first to perform a human clinical trial, in which treatment-naïve HIV infected individuals were vaccinated with protein or peptides pulsed autologous DCs [107]. The vaccine was well tolerated and resulted in an increase of HIV specific CD8+ T cell response in 3 out of 6 individuals, but no effect on viral load was observed. Three other clinical trials tested peptides pulsed DCs [101, 105, 106]. In two of these trials an increase in HIV-specific

**5.3. HIV-1 antigen loaded dendritic cells tested in clinical trails** 

infect new T cells by promoting tolerance of T cells to HIV-1 [157].

proliferative capacity of the activated CD8+ T cells.

suppressors of cytokine signaling (SOCS)-1 [98, 151].



"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 43

directly vaccinate HAART-treated patient or *ex vivo* load autologous DCs. The results obtained did not demonstrate clear differences in the immunological or virological outcome and a control group was lacking [102]. In a very recent study, DNA encoding HIV proteins was complexed with polymers and topically administrated with a patch method named DermaPrep [174]. The results showed that vaccination induced new HIV-specific immune responses in HAART treated patients. Moreover, a better control of viral replication during treatment interruption was observed [167]. DCs loaded with mRNA have also been tested in clinical trials. Routy *et al.* reported the results of a trial in which HAART-treated patients received autologous DCs *ex vivo* electroporated with mRNA encoding autologous HIV sequences and CD40L [173]. This pilot study showed CD8+ T cells specific for the presented HIV antigen were preferentially targeted. Two other clinical trials employing autologous DCs electroporated *ex vivo* with mRNA encoding Tat, Rev, Nef and Gag were recently concluded [175, 176]. Both studies showed that vaccine-specific CD4+ and CD8+ T cell responses were enhanced. In one study vaccinated individuals interrupted treatment. Six

One should keep in mind, however, that *ex vivo* manipulation of DCs is a complex personalized vaccination procedure, which prevents its accessibility to large numbers of patients. Therefore, there is a constant need for new approaches that could target DCs directly *in vivo*. In the following paragraphs we will discuss various non-viral delivery approaches which promote antigen uptake by DCs and induce potent immune responses.

Even though viral vectors are generally considered more efficient, non-viral delivery vehicles receive increasing attention since they are safer, more versatile, easier to prepare and hence more accessible for up-scaling [178]. In addition, they allow delivery of larger quantities of antigens. Importantly, encapsulation protects antigens from their environment and therefore permits a prolonged release of antigens in tissues or particular cells [179]. The usual size of particulate vaccines, ranging from a few hundred nanometers to a few microns, is ideal for uptake by DCs [180]. Moreover, the nature of non-viral carriers allows their functionalization with moieties permitting specific targeting to DCs. Additionally they allow co-delivery of immunostimulatory molecules which can direct the immune system toward the humoral or cellular arm [181]. The simultaneous delivery of antigens and immune-stimulators to the same DC is a feature which has been reported to significantly augment the strength of the induced adaptive immune responses [182- 184]. In the following paragraphs we will discuss different lipid- and polymer-based carrier systems employed to deliver proteins and nucleic acids relevant for HIV-specific

Peptides, proteins or nucleic acids (DNA or RNA) have been used as a source of antigen in the majority of therapeutic vaccination strategies. Each of them comes with specific

out of 17 individuals remained off therapy 96 weeks after cessation [175].

**6. The way forward: Antigen delivery by non-viral carriers** 

immunotherapy (**table 3**).

**6.1. Choice of antigen** 

**Table 2.** Overview of therapeutic DC-based clinical trials.

Nucleic acids are also used as a source of antigens in DC-based immunotherapies. Phase I and II clinical trials were conducted to evaluate the potential of a canarypox HIV-vaccine (ALVAC), containing DNA encoding Env, Gag as well as parts of Nef and Pol to either directly vaccinate HAART-treated patient or *ex vivo* load autologous DCs. The results obtained did not demonstrate clear differences in the immunological or virological outcome and a control group was lacking [102]. In a very recent study, DNA encoding HIV proteins was complexed with polymers and topically administrated with a patch method named DermaPrep [174]. The results showed that vaccination induced new HIV-specific immune responses in HAART treated patients. Moreover, a better control of viral replication during treatment interruption was observed [167]. DCs loaded with mRNA have also been tested in clinical trials. Routy *et al.* reported the results of a trial in which HAART-treated patients received autologous DCs *ex vivo* electroporated with mRNA encoding autologous HIV sequences and CD40L [173]. This pilot study showed CD8+ T cells specific for the presented HIV antigen were preferentially targeted. Two other clinical trials employing autologous DCs electroporated *ex vivo* with mRNA encoding Tat, Rev, Nef and Gag were recently concluded [175, 176]. Both studies showed that vaccine-specific CD4+ and CD8+ T cell responses were enhanced. In one study vaccinated individuals interrupted treatment. Six out of 17 individuals remained off therapy 96 weeks after cessation [175].

One should keep in mind, however, that *ex vivo* manipulation of DCs is a complex personalized vaccination procedure, which prevents its accessibility to large numbers of patients. Therefore, there is a constant need for new approaches that could target DCs directly *in vivo*. In the following paragraphs we will discuss various non-viral delivery approaches which promote antigen uptake by DCs and induce potent immune responses.

#### **6. The way forward: Antigen delivery by non-viral carriers**

Even though viral vectors are generally considered more efficient, non-viral delivery vehicles receive increasing attention since they are safer, more versatile, easier to prepare and hence more accessible for up-scaling [178]. In addition, they allow delivery of larger quantities of antigens. Importantly, encapsulation protects antigens from their environment and therefore permits a prolonged release of antigens in tissues or particular cells [179]. The usual size of particulate vaccines, ranging from a few hundred nanometers to a few microns, is ideal for uptake by DCs [180]. Moreover, the nature of non-viral carriers allows their functionalization with moieties permitting specific targeting to DCs. Additionally they allow co-delivery of immunostimulatory molecules which can direct the immune system toward the humoral or cellular arm [181]. The simultaneous delivery of antigens and immune-stimulators to the same DC is a feature which has been reported to significantly augment the strength of the induced adaptive immune responses [182- 184]. In the following paragraphs we will discuss different lipid- and polymer-based carrier systems employed to deliver proteins and nucleic acids relevant for HIV-specific immunotherapy (**table 3**).

#### **6.1. Choice of antigen**

42 Immunodeficiency

**Form of antigen** 

> DCs pulsed with HLA A\*0201 binding epitopes in Gag, Pol and Env

> DCs pulsed with HLA A\*0201 binding epitopes in Gag, Pol, Env, Vpu and Vif and Th epitopes

ALVAC-Remune HAART treated

in Gag and Env

**DNA** ALVAC pulsed DCs vs AlVAC alone

**mRNA** *Ex vivo* electroporated

DCs with mRNA encoding CD40L and autologous HIV proteins (Gag, Ref, Nef, Vpr)

*Ex vivo* electroporated DCs with mRNA and autologous HIV proteins (Gag, Tat, Rev, Nef)

*Ex vivo* electroporated DCs with mRNA and autologous HIV proteins

**Table 2.** Overview of therapeutic DC-based clinical trials.

(Tat, Rev, Nef)

**Vaccine strategy Subjects Clinical trial outcome** 

HAART treated patients (n=18)

HAART treated patients followed by therapy interruption (n=29)

patients followed by therapy interruption (n=48)

HAART treated patients (n=10)

HAART treated patients (n=6)

HAART treated patients followed by therapy interruption (n=17)

Nucleic acids are also used as a source of antigens in DC-based immunotherapies. Phase I and II clinical trials were conducted to evaluate the potential of a canarypox HIV-vaccine (ALVAC), containing DNA encoding Env, Gag as well as parts of Nef and Pol to either

Untreated patients (n= 12) Increase in CTL responses against

No differences in T cell responses against HIV antigens. Did not lower VL setpoint after therapy

No lowering of viral setpoint after vaccination, but VL rebound

HIV specific proliferative

targeted to CD8+ T cells. Correlation with viral control during therapy interruption.

with CD4+ and CD8+

Autologous CD8+ T cells inhibited HIV superinfection in

[109]

*vitro*. [176]

immune responses preferentially

Breadth of IFN-γ response and Tcell proliferation were correlated

polyfunctional T-cell responses.

Induced or enhanced CD4+ and CD8+ T cell responses specific for the vaccine antigens. [175]

vaccine epitopes. [101]

Generation of new T cell responses despite high viral

loads. [106]

interruption. [102]

was delayed. [177]

Peptides, proteins or nucleic acids (DNA or RNA) have been used as a source of antigen in the majority of therapeutic vaccination strategies. Each of them comes with specific characteristics in terms of safety, stability, potential to cover antigenic variability, requirements for delivery and nature of the immune response induced [185]. Recombinant proteins or peptides utilized as subunit vaccines are safe and simple forms of antigens. However, their production at clinical grade quality is very expensive. As a consequence, only one or a few antigenic variants can be produced at an affordable price. Moreover, they are susceptible to pre-mature proteolytic degradation. Since DCs recognize them as exogenous antigens, they are preferentially presented in a MHC class II context [143] and to a lesser extent onto MHC I molecules via cross-presentation [180]. Therefore, they will not induce strong and broad CD8+ T cell responses, which are considered as a prerequisite for a therapeutic vaccine. These limitations can partially be resolved by their encapsulation. Encapsulation protects proteins and peptides from being prematurely degraded by proteolytic enzymes. Additionally, particulate antigen delivery favors cross-presentation thereby enhancing CD8+ T cell responses [186, 187]. In the context of HIV immunotherapy, however, the use of proteins as antigens is not ideal since HIV generates escape variants during the course of infection which remain present as a latent reservoir in cells [188]. With the technology available, it is not feasible to produce hundreds of variants of the same protein and include them in a therapeutic vaccine.

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 45

to pDNA, mRNA-based delivery may hold several advantages. First of all, mRNA is easier to engineer; there is no need for specific promoters and terminators to be present in the construct. Secondly, the synthesis of proteins encoded by mRNA is transient, which ensures a controlled antigen exposure [191]. Thirdly, in contrast to pDNA, mRNA does not need to cross the nuclear membrane to be effective and therefore offers the possibility to produce proteins in slow or non-dividing cells [192]. Furthermore, the use of mRNA excludes the risk of integration into the cell genome, eliminating possible insertional mutagenesis [189, 193]. For years, the application of mRNA has been hampered by a general believe that it is too labile to guarantee sufficient protein expression. Nowadays, however, mRNA vaccination strategies are being thoroughly investigated in the field of allergy [194, 195], cancer [193, 196-198] and HIV [109, 170, 171] immunotherapy. These studies mostly rely on the *ex vivo* electroporation of dendritic cells. We and others focus on complexing mRNA with cationic lipids and polymers (**figure 4**), which could protect mRNA. Moreover, we believe that complexing mRNA with cationic carriers might hold potential for their application *in vivo*. This approach could serve as

**Figure 4.** Internalisation and intracellular trafficking of mRNA/cationic carrier complexes. Negatively charged mRNA is complexed with cationic carriers (lipid or polymer based). After endocytosis, complexes end up in endosomes, from where they should release their cargo mRNA into the cytoplasm to enable translation by ribosomes .Translated protein is either secreted or can directly be processed via

proteaseome to be presented with MHC class I. Secreted protein can be taken up and promote

presentation in a MHC class II way after cleavage in the endolysosomes.

an alternative for laborious *ex vivo* loading of DCs.


**Table 3.** Advantages and disadvantages of different non-viral delivery systems.

Nucleic acids, such a plasmid DNA (pDNA) or messenger RNA (mRNA), encoding viral proteins, can more easily cover the wide range of viral quasi species [189, 190]. As compared to pDNA, mRNA-based delivery may hold several advantages. First of all, mRNA is easier to engineer; there is no need for specific promoters and terminators to be present in the construct. Secondly, the synthesis of proteins encoded by mRNA is transient, which ensures a controlled antigen exposure [191]. Thirdly, in contrast to pDNA, mRNA does not need to cross the nuclear membrane to be effective and therefore offers the possibility to produce proteins in slow or non-dividing cells [192]. Furthermore, the use of mRNA excludes the risk of integration into the cell genome, eliminating possible insertional mutagenesis [189, 193]. For years, the application of mRNA has been hampered by a general believe that it is too labile to guarantee sufficient protein expression. Nowadays, however, mRNA vaccination strategies are being thoroughly investigated in the field of allergy [194, 195], cancer [193, 196-198] and HIV [109, 170, 171] immunotherapy. These studies mostly rely on the *ex vivo* electroporation of dendritic cells. We and others focus on complexing mRNA with cationic lipids and polymers (**figure 4**), which could protect mRNA. Moreover, we believe that complexing mRNA with cationic carriers might hold potential for their application *in vivo*. This approach could serve as an alternative for laborious *ex vivo* loading of DCs.

44 Immunodeficiency

**Delivery vehicle** 

PLA-PLGA particles

Polyelctrolyte capsules

Polyplexes Nucleic

Liposomes Proteins,

Lipoplexes Nucleic

protein and include them in a therapeutic vaccine.

**Form of antigen** 

Proteins, peptides

acids

peptides, nucleic acids

acids

characteristics in terms of safety, stability, potential to cover antigenic variability, requirements for delivery and nature of the immune response induced [185]. Recombinant proteins or peptides utilized as subunit vaccines are safe and simple forms of antigens. However, their production at clinical grade quality is very expensive. As a consequence, only one or a few antigenic variants can be produced at an affordable price. Moreover, they are susceptible to pre-mature proteolytic degradation. Since DCs recognize them as exogenous antigens, they are preferentially presented in a MHC class II context [143] and to a lesser extent onto MHC I molecules via cross-presentation [180]. Therefore, they will not induce strong and broad CD8+ T cell responses, which are considered as a prerequisite for a therapeutic vaccine. These limitations can partially be resolved by their encapsulation. Encapsulation protects proteins and peptides from being prematurely degraded by proteolytic enzymes. Additionally, particulate antigen delivery favors cross-presentation thereby enhancing CD8+ T cell responses [186, 187]. In the context of HIV immunotherapy, however, the use of proteins as antigens is not ideal since HIV generates escape variants during the course of infection which remain present as a latent reservoir in cells [188]. With the technology available, it is not feasible to produce hundreds of variants of the same

**Advantages Disadvantages** 

Harsh preparation process, expensive to

Strong electrostatic interactions may hamper release of nucleic acids from the

Poorly immunogenic

Might aggregation in the presence of serum

upscale.

carrier

Not (yet) FDAapproved

Proteins FDA approved, induction of specific

number of bilayers.

**Table 3.** Advantages and disadvantages of different non-viral delivery systems.

antibodies and Th1 cellular responses

Protect nucleic acids, facilitate escape from the endosomal compartment.

Good protection of antigens, facilitate intracellular uptake. Induction of both Th1 and Th2 responses.

Protect nucleic acids, facilitate escape from the endosomal compartment.

Nucleic acids, such a plasmid DNA (pDNA) or messenger RNA (mRNA), encoding viral proteins, can more easily cover the wide range of viral quasi species [189, 190]. As compared

Induction of both Th1 and Th2 responses. Easy to tailor with immunostimulators. Stability and release kinetics correlate with a

**Figure 4.** Internalisation and intracellular trafficking of mRNA/cationic carrier complexes. Negatively charged mRNA is complexed with cationic carriers (lipid or polymer based). After endocytosis, complexes end up in endosomes, from where they should release their cargo mRNA into the cytoplasm to enable translation by ribosomes .Translated protein is either secreted or can directly be processed via proteaseome to be presented with MHC class I. Secreted protein can be taken up and promote presentation in a MHC class II way after cleavage in the endolysosomes.

#### **6.2. Polymer-based antigen delivery systems**

Various polymers have been used to prepare nano- and micro- particles for antigen delivery. Here, we will focus on polymers that have shown to be promising in terms of immunotherapeutic vaccination.

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 47

delivered in the same particle to DCs inducing maturation and stimulation of HIV-specific responses both *in vitro* and *in vivo* [184]. PeMCs were also used for the delivery of Gagpeptides to APCs. These APCs could activate SIV-specific T cells in an *ex vivo* non-human primate model [217]. Immune responses could be further improved by sensitizing the PeMCs to enzymatic or reductive degradation upon cellular uptake, thereby assuring

PeMCs have been also employed to deliver pDNA [213, 220]. When considering their use for the delivery of mRNA, however, one should make sure that the preparation process is

**Figure 5.** Preparation of polyelectrolyte microcapsule [186]. (a) In the first step an antigen (yellow) of interest is mixed with colloid nanoparticles (grey) to form a core template. (b) In the next step a layer of negatively charged polymer is deposited (blue). The non-adsorbed polymer is removed by washing. This is followed by applying a positively charged polymer (red) and another washing step. (c) When

Polyethyleneimine (PEI) has been extensively used to deliver pDNA and siRNA to cells [221-223]. PEI consists of repeating units that contain two carbon atoms and a protonatable nitrogen atom. It exists in a linear or branched conformation (**figure 6**), both appearing in a broad range of molecular weights. PEI can efficiently bind pDNA to form so-called polyplexes. The linear form has been shown to release complexed pDNA more easily than the more stable branched form and thus results in higher transfection efficiencies [224]. The net positive charge of the polyplexes promotes their adhesion to the overall negative charge of the cellular membrane and facilitates their uptake. The protonatable units of PEI have a buffering capacity resulting in an influx of hydrogen ions into the endosomes upon polyplex uptake. Due to osmotic pressure thus building up, the endosomes are disrupted resulting in complex release into the cytosol [222, 225]. This mechanism of escape from the endosomes is

The group of Lisziewicz developed a therapeutic HIV vaccine, DermaVir, consisting of PEImannose complexed with pDNA encoding several HIV-1 antigens [227]. The vaccine was administered using a patch (DermaVir Patch) and was meant to target Langerhans cells (LCs) [174]. The vaccine induced specific and long lasting immune responses resulting in reduced viral load in SIV-infected macaques [228]. The safety of the vaccine formulation and of the delivery method was demonstrated in a phase I clinical trial in humans. A phase II

the desired number of polyelectrolyte layers is obtained, the core is dissolved.

*6.2.3. Polyethyleneimine-based polyplexes* 

called the proton sponge mechanism [226].

release of its cargo [218, 219].

rigorously RNase-free, which might present a challenge.

#### *6.2.1. Polymers based on lactic acid (PLA) and glycolic acid (PLGA)*

The most studied polymers for antigen delivery in the context of vaccination are the biodegradable poly(D,L-lactide) (PLA) and poly(D,L-lactic-*co*-glycolic acid) (PLGA), which have been FDA approved for human use. These particles have mainly been evaluated for their potential to deliver proteins and peptides to DCs or macrophages with the aim to induce CD8+ cytotoxic T cell immune responses in the context of prophylactic vaccination. It has been demonstrated that PLGA particles are phagocytosed by APCs [199] and ensure prolonged antigen presentation by DCs [200]. Whereas empty PLGA particles do not influence the maturation status of DCs [201, 202], DC activation can be induced, if the PLGA polymer is employed to encapsulate TLR ligands (poly I:C, MPLA) or surface loaded with anti-CD40 antibodies clear activation of DCs was observed [203-205]. PLA particles carrying p24 protein have been shown to induce both mucosal antibody production as well as CTL responses [206, 207].

The main drawback of these polymers is that some antigens tend to aggregate during the encapsulation process. Moreover, the exposure of proteins to organic solvents, required to dissolve the polymer, makes them highly susceptible to denaturation leading to the loss of antigenic epitope recognition [208]. This can partially be overcome by adsorption of proteins on the particle surface [209, 210]. Additionally, a rather expensive up-scaling process and clean-up procedure to ensure sterile production constitute difficult obstacles [211]. Due to the harsh preparation process and the hydrophobic nature of PLGA and PLA, these carriers are not suitable for nucleic acids delivery [212-214].

#### *6.2.2. Polyelectrolyte microcapsules*

Polyelectrolyte microcapsules (PeMCs) fabricated using a so-called layer-by-layer (LbL) technology are a relatively novel class of particles [215]. The process of their preparation is less harsh as compared to that of PLGA/PLA particles. A template containing an antigen and colloid nanoparticles is used as a sacrificial core. This core is coated with several bilayers of polymers of opposite charges. At the end of the procedure the template core is dissolved (**figure 5**). Since the encapsulation process is performed in a purely aqueous environment, minimal stress for a protein antigen is ensured. With appropriate choice of polymers, these particles can be fully biodegradable. De Koker *et al.* used ovalbumin as a model antigen and showed the benefit of antigen encapsulation in PeMCs to stimulate antigen presentation to T cells by murine bone marrow-derived DCs [186, 216].

PeMCs containing p24 and poly I:C (TLR-3 ligand) have been shown to be promising as an HIV-1 immunotherapeutic vaccine. Both antigen and maturation stimulus could be delivered in the same particle to DCs inducing maturation and stimulation of HIV-specific responses both *in vitro* and *in vivo* [184]. PeMCs were also used for the delivery of Gagpeptides to APCs. These APCs could activate SIV-specific T cells in an *ex vivo* non-human primate model [217]. Immune responses could be further improved by sensitizing the PeMCs to enzymatic or reductive degradation upon cellular uptake, thereby assuring release of its cargo [218, 219].

PeMCs have been also employed to deliver pDNA [213, 220]. When considering their use for the delivery of mRNA, however, one should make sure that the preparation process is rigorously RNase-free, which might present a challenge.

**Figure 5.** Preparation of polyelectrolyte microcapsule [186]. (a) In the first step an antigen (yellow) of interest is mixed with colloid nanoparticles (grey) to form a core template. (b) In the next step a layer of negatively charged polymer is deposited (blue). The non-adsorbed polymer is removed by washing. This is followed by applying a positively charged polymer (red) and another washing step. (c) When the desired number of polyelectrolyte layers is obtained, the core is dissolved.

#### *6.2.3. Polyethyleneimine-based polyplexes*

46 Immunodeficiency

**6.2. Polymer-based antigen delivery systems** 

are not suitable for nucleic acids delivery [212-214].

*6.2.2. Polyelectrolyte microcapsules* 

*6.2.1. Polymers based on lactic acid (PLA) and glycolic acid (PLGA)* 

immunotherapeutic vaccination.

responses [206, 207].

Various polymers have been used to prepare nano- and micro- particles for antigen delivery. Here, we will focus on polymers that have shown to be promising in terms of

The most studied polymers for antigen delivery in the context of vaccination are the biodegradable poly(D,L-lactide) (PLA) and poly(D,L-lactic-*co*-glycolic acid) (PLGA), which have been FDA approved for human use. These particles have mainly been evaluated for their potential to deliver proteins and peptides to DCs or macrophages with the aim to induce CD8+ cytotoxic T cell immune responses in the context of prophylactic vaccination. It has been demonstrated that PLGA particles are phagocytosed by APCs [199] and ensure prolonged antigen presentation by DCs [200]. Whereas empty PLGA particles do not influence the maturation status of DCs [201, 202], DC activation can be induced, if the PLGA polymer is employed to encapsulate TLR ligands (poly I:C, MPLA) or surface loaded with anti-CD40 antibodies clear activation of DCs was observed [203-205]. PLA particles carrying p24 protein have been shown to induce both mucosal antibody production as well as CTL

The main drawback of these polymers is that some antigens tend to aggregate during the encapsulation process. Moreover, the exposure of proteins to organic solvents, required to dissolve the polymer, makes them highly susceptible to denaturation leading to the loss of antigenic epitope recognition [208]. This can partially be overcome by adsorption of proteins on the particle surface [209, 210]. Additionally, a rather expensive up-scaling process and clean-up procedure to ensure sterile production constitute difficult obstacles [211]. Due to the harsh preparation process and the hydrophobic nature of PLGA and PLA, these carriers

Polyelectrolyte microcapsules (PeMCs) fabricated using a so-called layer-by-layer (LbL) technology are a relatively novel class of particles [215]. The process of their preparation is less harsh as compared to that of PLGA/PLA particles. A template containing an antigen and colloid nanoparticles is used as a sacrificial core. This core is coated with several bilayers of polymers of opposite charges. At the end of the procedure the template core is dissolved (**figure 5**). Since the encapsulation process is performed in a purely aqueous environment, minimal stress for a protein antigen is ensured. With appropriate choice of polymers, these particles can be fully biodegradable. De Koker *et al.* used ovalbumin as a model antigen and showed the benefit of antigen encapsulation in PeMCs to stimulate

PeMCs containing p24 and poly I:C (TLR-3 ligand) have been shown to be promising as an HIV-1 immunotherapeutic vaccine. Both antigen and maturation stimulus could be

antigen presentation to T cells by murine bone marrow-derived DCs [186, 216].

Polyethyleneimine (PEI) has been extensively used to deliver pDNA and siRNA to cells [221-223]. PEI consists of repeating units that contain two carbon atoms and a protonatable nitrogen atom. It exists in a linear or branched conformation (**figure 6**), both appearing in a broad range of molecular weights. PEI can efficiently bind pDNA to form so-called polyplexes. The linear form has been shown to release complexed pDNA more easily than the more stable branched form and thus results in higher transfection efficiencies [224]. The net positive charge of the polyplexes promotes their adhesion to the overall negative charge of the cellular membrane and facilitates their uptake. The protonatable units of PEI have a buffering capacity resulting in an influx of hydrogen ions into the endosomes upon polyplex uptake. Due to osmotic pressure thus building up, the endosomes are disrupted resulting in complex release into the cytosol [222, 225]. This mechanism of escape from the endosomes is called the proton sponge mechanism [226].

The group of Lisziewicz developed a therapeutic HIV vaccine, DermaVir, consisting of PEImannose complexed with pDNA encoding several HIV-1 antigens [227]. The vaccine was administered using a patch (DermaVir Patch) and was meant to target Langerhans cells (LCs) [174]. The vaccine induced specific and long lasting immune responses resulting in reduced viral load in SIV-infected macaques [228]. The safety of the vaccine formulation and of the delivery method was demonstrated in a phase I clinical trial in humans. A phase II clinical trial started in 2009 and aims at evaluating immunogenicity and efficacy of the vaccine in treatment-naïve and HAART-treated patients [229, 230].

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 49

has been reported recently that mice immunized with Gag protein encapsulated in liposomes functionalized with lipid A induced both humoral and cellular immunity [236]. Another strategy to increase immunogenic potential of liposomal formulations involves the use of so-called lipopeptides. These structures consist of lipids linked to peptides that contain potential CD4+ and CD8+ T cell epitopes. These vaccines aim at the induction of strong T cell responses in HIV-1 infected patients [237-239]. Currently, a lipopeptide-pulsed DC vaccine is under evaluation in a phase I/II clinical trial in HAART-treated patients

The combination of positively charged lipids and negatively charged nucleic acids results in spontaneous formation of complexes called lipoplexes (**figure 7c**). These systems typically consist of two lipid species: a cationic lipid (such as DOTAP - 1,2-dioleoyl-3 trimethylammonium-propane) and a helper lipid with long unsaturated fatty acid chains (such as DOPE - dioleoylphosphatidylethanolamine) (**figure 7**). New cationic lipids are being synthesized almost daily aiming at introducing additional positive charges and reducing toxicity. Helper lipids are introduced to facilitate endosomal escape of these

The positive charge of lipoplexes promotes their cellular uptake, which was found to occur via clathrin-dependent and independent endocytosis [243-245]. The route of lipoplex uptake is determined by their size, chemical nature of the lipids and the cell line. After being internalised, the lipoplexes are located in endosomes. Escape from these compartments probably occurs via a mechanism proposed by Xu and Szoka [246]. It implies formation of neutral molecular pairs of lipoplex-derived cationic lipids and negatively charged phospholipids present in the endosomal membrane, eventually leading to release of the

**Figure 7.** Chemical structures of DOTAP, cationic lipid, and DOPE, neutral helper lipid, used to form lipoplexes [213, 242]. DOTAP (a) and DOPE (b) contain a hydrophilic head group, a glycerol linker and two hydrophobic fatty acid chains. (c) Schematic representation of a lipoplex containing nucleic acids (blue) stabilized by bilayer of lipids: hydrophilic groups (red), hydrophobic fatty acid chain

chronically infected with HIV-1 [240].

*6.3.2 Cationic lipid based lipoplexes* 

complexes [241].

(grey).

nucleic acid into the cytoplasm.

**Figure 6.** Structures of polyethyleneimine: (a) Linear backbone. (b) Branched backbone.

The main drawback of using PEI is its toxicity and non-specific interactions with cellular compartments. Another challenge is the aggregation of polyplexes in the presence of serum.

Rejman *et al.* reported that complexes made of mRNA and cationic lipids are much more efficient in transfecting different cell lines than PEI polyplexes. The authors suggest that this was likely due to the strong affinity of the polymer to mRNA impeding the release of the nucleic acid from the complexes. [191]. Bettinger *et al*. tested the potency of different protein derivatives for the delivery of mRNA. They demonstrated that complexing mRNA with short peptides results in better transfection efficiencies, likely due to a weaker electrostatic interaction with the nucleic acid. It should be noted, however, that the expression levels obtained were in general very low [192].

#### **6.3. Lipid-based antigen delivery systems**

#### *6.3.1. Liposomes*

Liposomes are spherical entities consisting of a phospholipid bilayer and an aqueous inner compartment. They have been used for drug delivery to treat cancer and infectious diseases. Until now, few liposomal formulations reached the pharmaceutical market of the U.S.A. Liposomes have been also employed to deliver antigens [231]. Given the liposome structure, the antigen can be encapsulated in its core (hydrophilic molecules) or accommodated within the lipid bilayer (hydrophobic molecules) [232]. It has been demonstrated that the lipid composition determines the immunogenicity of liposomes. For example, the incorporation of cationic lipids has been shown to elicit elevated CTL responses compared to neutral or anionic lipids [233]. Liposomal particles can be modified with specific ligands or antibodies to improve the uptake or to enhance/skew the immune response. Virosomes or virus like particles that consist of functional viral envelope proteins, anchored in a lipid membrane, have proven to be promising vaccine candidates [234]. Importantly, antigenic proteins encapsulated in liposomes can elicit both MHC I and II mediated immune responses as demonstrated by Zheng *et al.* [235]. These authors showed that HIV-1 Gag, Pol and Env proteins delivered to DCs by cationic liposomes induced stimulation of HIV-1 specific CD8+ T cell responses. This was not observed when DCs were pulsed with the soluble proteins. It has been reported recently that mice immunized with Gag protein encapsulated in liposomes functionalized with lipid A induced both humoral and cellular immunity [236]. Another strategy to increase immunogenic potential of liposomal formulations involves the use of so-called lipopeptides. These structures consist of lipids linked to peptides that contain potential CD4+ and CD8+ T cell epitopes. These vaccines aim at the induction of strong T cell responses in HIV-1 infected patients [237-239]. Currently, a lipopeptide-pulsed DC vaccine is under evaluation in a phase I/II clinical trial in HAART-treated patients chronically infected with HIV-1 [240].

#### *6.3.2 Cationic lipid based lipoplexes*

48 Immunodeficiency

clinical trial started in 2009 and aims at evaluating immunogenicity and efficacy of the

The main drawback of using PEI is its toxicity and non-specific interactions with cellular compartments. Another challenge is the aggregation of polyplexes in the presence of serum. Rejman *et al.* reported that complexes made of mRNA and cationic lipids are much more efficient in transfecting different cell lines than PEI polyplexes. The authors suggest that this was likely due to the strong affinity of the polymer to mRNA impeding the release of the nucleic acid from the complexes. [191]. Bettinger *et al*. tested the potency of different protein derivatives for the delivery of mRNA. They demonstrated that complexing mRNA with short peptides results in better transfection efficiencies, likely due to a weaker electrostatic interaction with the nucleic acid. It should be noted, however, that the expression levels

Liposomes are spherical entities consisting of a phospholipid bilayer and an aqueous inner compartment. They have been used for drug delivery to treat cancer and infectious diseases. Until now, few liposomal formulations reached the pharmaceutical market of the U.S.A. Liposomes have been also employed to deliver antigens [231]. Given the liposome structure, the antigen can be encapsulated in its core (hydrophilic molecules) or accommodated within the lipid bilayer (hydrophobic molecules) [232]. It has been demonstrated that the lipid composition determines the immunogenicity of liposomes. For example, the incorporation of cationic lipids has been shown to elicit elevated CTL responses compared to neutral or anionic lipids [233]. Liposomal particles can be modified with specific ligands or antibodies to improve the uptake or to enhance/skew the immune response. Virosomes or virus like particles that consist of functional viral envelope proteins, anchored in a lipid membrane, have proven to be promising vaccine candidates [234]. Importantly, antigenic proteins encapsulated in liposomes can elicit both MHC I and II mediated immune responses as demonstrated by Zheng *et al.* [235]. These authors showed that HIV-1 Gag, Pol and Env proteins delivered to DCs by cationic liposomes induced stimulation of HIV-1 specific CD8+ T cell responses. This was not observed when DCs were pulsed with the soluble proteins. It

vaccine in treatment-naïve and HAART-treated patients [229, 230].

**Figure 6.** Structures of polyethyleneimine: (a) Linear backbone. (b) Branched backbone.

obtained were in general very low [192].

*6.3.1. Liposomes* 

**6.3. Lipid-based antigen delivery systems** 

The combination of positively charged lipids and negatively charged nucleic acids results in spontaneous formation of complexes called lipoplexes (**figure 7c**). These systems typically consist of two lipid species: a cationic lipid (such as DOTAP - 1,2-dioleoyl-3 trimethylammonium-propane) and a helper lipid with long unsaturated fatty acid chains (such as DOPE - dioleoylphosphatidylethanolamine) (**figure 7**). New cationic lipids are being synthesized almost daily aiming at introducing additional positive charges and reducing toxicity. Helper lipids are introduced to facilitate endosomal escape of these complexes [241].

The positive charge of lipoplexes promotes their cellular uptake, which was found to occur via clathrin-dependent and independent endocytosis [243-245]. The route of lipoplex uptake is determined by their size, chemical nature of the lipids and the cell line. After being internalised, the lipoplexes are located in endosomes. Escape from these compartments probably occurs via a mechanism proposed by Xu and Szoka [246]. It implies formation of neutral molecular pairs of lipoplex-derived cationic lipids and negatively charged phospholipids present in the endosomal membrane, eventually leading to release of the nucleic acid into the cytoplasm.

**Figure 7.** Chemical structures of DOTAP, cationic lipid, and DOPE, neutral helper lipid, used to form lipoplexes [213, 242]. DOTAP (a) and DOPE (b) contain a hydrophilic head group, a glycerol linker and two hydrophobic fatty acid chains. (c) Schematic representation of a lipoplex containing nucleic acids (blue) stabilized by bilayer of lipids: hydrophilic groups (red), hydrophobic fatty acid chain (grey).

Lipoplexes are mainly used to transfect primary cells or cell lines *in vitro*. However, since the rise of DNA vaccines, cationic lipids are considered as a versatile method to replace the hazardous viral vectors. In mice DNA lipoplexes have been shown to induce antitumor activity [247] and both antibody and Th1 cellular responses against hepatitis C virus [248, 249]. In the late eighties the first transfection with mRNA-lipoplexes was performed [250]. Generally it is established that mRNA transfection using non-viral cationic carriers is more efficient with cationic lipids than with cationic polymers [191, 192, 251, 252]. For both types of mRNA complexes the onset of protein expression was faster, but also lasted shorter than that produced by pDNA-complexes [191, 252]. The potential of mRNA-lipoplexes as an anticancer or influenza vaccine dates back to the nineties [253, 254]. Since then most anti-cancer and anti-virus immunotherapies have been focused on the *ex vivo* loading of DCs mainly by means of electroporation [109, 196, 198, 255, 256].

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 51

receptors present on DCs and macrophages. This has been shown to increase transfection efficiency of DNA-based vaccines [269, 270], antigen presentation following protein delivery [271] and the induction of cellular T cell responses in cancer and HIV immunotherapeutic strategies using lipoplexes and polyplexes [228, 270]. Another way of targeting is the use of ligands or antibodies directed against DEC-205 or DC-SIGN, which have shown potential to improve antigen uptake by immature and mature DCs [272-275]. It should be kept in mind, however, that attaching the desired ligand or protein is not always a straightforward process. The engraftment of chelator lipids on the surface of liposomes or lipoplexes, such as histidine tags, is crucial to ensure the functionality of the coupled target molecule [276-278]. In this context, the use of nanobodies directed against DCs could be of particular interest as they are mostly generated with a histidine tag for purification purposes. Alternatively,

ligands could also be linked to liposomes via palmitoylation of the ligand [279].

positively influence the uptake by DCs [280].

and both humoral and cellular responses were induced.

In summary, targeting of particles may not only enhance efficacy but also the specificity of interaction with the surface receptors on DCs [179]. Moreover, coupling of specific adjuvants (e.g. TLR ligands), with the aim to promote the desired immune response, can also

**6.5. Co-delivery of antigens and adjuvants to improve immunogenicity of DCs** 

Although particulate antigen delivery improves antigen uptake and presentation by DCs, most particles are not immunogenic by themselves. This offers a possibility to use a specific adjuvant that can skew towards the desired type of immune responses [187]. Despite some positive results in animal models, one should be aware of the toxicity of potential immunomodulators in humans, that jeopardizes their clinical use [281]. Aluminum salts (referred to as alum) are the oldest and most widely used adjuvants for human vaccines. It has been shown that antigens can be precipitated with alum to form colloid particles, creating a depot effect after vaccination, that elicits strong humoral immune responses [282]. These results, together with further observations clearly showed that the physical linkage of antigen and immunomodulator is crucial to induce strong immune responses [182, 183, 283]. Immunostimulating complexes (ISCOMs) are another interesting delievery system. It is a sort of liposomal delivery vehicle with a built-in adjuvant. They are composed of a protein antigen, phosopholipids and the saponin Quil A adjuvant, derived from the bark of the *Quillaja saponaria*, a South American tree. It has been demonstrated that the use of ISCOMs improves CTL responses of influenza virus based vaccines [284, 285]. ISCOM-based vaccines have been tested in animal models and in clinical trials against cancer [286] and viral infections [287-289] [290, 291]. In all studies satisfactory safety and tolerance were shown

Several studies have reported the use of ISCOMs as a system to deliver HIV or SIV antigens. In non-human primate models, incorporation of HIV or SIV peptides into ISCOMs has been shown to induce protective immunity [288, 292, 293]. Moreover, studies in mice demonstrated that ISCOMs can be used to elicit immune responses against HIV-1 antigens [287]. To generate mucosal immunity, Koopman and colleagues immunized rhesus

One of the most serious challenges of the use of lipoplexes is the extrapolation from the *in vitro* to the *in vivo* situation. Optimal transfection conditions are maintained *in vitro*, while *in vivo* lipoplexes encounter serum proteins causing their aggregation [257]. Depending on the target cells or tissue, this aggregation can either negatively or positively affect the uptake of the lipoplexes [258, 259]. Additional coating of the lipoplexes with polyethyleneglycol (PEG) can prevent the formation of aggregates [260], yet brings along other problems. Concerns have been raised that PEGylation of lipoplexes may be unfavorable in the context of vaccination as this could inhibit uptake of nanoparticles by APCs and negatively interfere with the release of complexes from the endosomal compartment [213]. However, a study performed by Singh and colleagues demonstrated that subcutaneous immunization of rabbits with PEGylated lipoplexes carrying a synthetic gp41 epitope of HIV-1 induced two times higher immune responses and prolonged persistence of antibodies than liposomes carrying epitopes without PEG moieties [261].

#### **6.4. Targeting of DCs**

Conceptually, particulate vaccines mimic the particulate nature of pathogens, including the size (nano- to micro-meter range), which facilitates their uptake by APCs. The actual size of the particles influences the uptake mechanism by APCs and the way of antigen presentation. Generally, larger particles are predominantly internalised via phagocytosis or macropinocytosis, while smaller particles are taken up by other endocytic mechanisms [262- 264]. Internalisation through phagocytosis is known to lead to antigen cross-presentation in DCs [265, 266]. This emphasizes the enhanced potential of particulate antigen delivery to induce cellular immune responses as compared to soluble antigens, which are poorly crosspresented and preferentially presented via the MHC class II pathway.

Most nano- or microparticles can be functionalized with ligands or antibodies directed against cell surface receptors to target specific tissues or cells [267]. DCs express lectin-like receptors such as mannose receptor, DEC-205 and DC-SIGN which are believed to be involved in the phagocytosis of pathogens [268]. One way of increasing or promoting particle uptake by DCs is the attachment of mannose groups recognized by the mannose receptors present on DCs and macrophages. This has been shown to increase transfection efficiency of DNA-based vaccines [269, 270], antigen presentation following protein delivery [271] and the induction of cellular T cell responses in cancer and HIV immunotherapeutic strategies using lipoplexes and polyplexes [228, 270]. Another way of targeting is the use of ligands or antibodies directed against DEC-205 or DC-SIGN, which have shown potential to improve antigen uptake by immature and mature DCs [272-275]. It should be kept in mind, however, that attaching the desired ligand or protein is not always a straightforward process. The engraftment of chelator lipids on the surface of liposomes or lipoplexes, such as histidine tags, is crucial to ensure the functionality of the coupled target molecule [276-278]. In this context, the use of nanobodies directed against DCs could be of particular interest as they are mostly generated with a histidine tag for purification purposes. Alternatively, ligands could also be linked to liposomes via palmitoylation of the ligand [279].

50 Immunodeficiency

means of electroporation [109, 196, 198, 255, 256].

carrying epitopes without PEG moieties [261].

**6.4. Targeting of DCs** 

Lipoplexes are mainly used to transfect primary cells or cell lines *in vitro*. However, since the rise of DNA vaccines, cationic lipids are considered as a versatile method to replace the hazardous viral vectors. In mice DNA lipoplexes have been shown to induce antitumor activity [247] and both antibody and Th1 cellular responses against hepatitis C virus [248, 249]. In the late eighties the first transfection with mRNA-lipoplexes was performed [250]. Generally it is established that mRNA transfection using non-viral cationic carriers is more efficient with cationic lipids than with cationic polymers [191, 192, 251, 252]. For both types of mRNA complexes the onset of protein expression was faster, but also lasted shorter than that produced by pDNA-complexes [191, 252]. The potential of mRNA-lipoplexes as an anticancer or influenza vaccine dates back to the nineties [253, 254]. Since then most anti-cancer and anti-virus immunotherapies have been focused on the *ex vivo* loading of DCs mainly by

One of the most serious challenges of the use of lipoplexes is the extrapolation from the *in vitro* to the *in vivo* situation. Optimal transfection conditions are maintained *in vitro*, while *in vivo* lipoplexes encounter serum proteins causing their aggregation [257]. Depending on the target cells or tissue, this aggregation can either negatively or positively affect the uptake of the lipoplexes [258, 259]. Additional coating of the lipoplexes with polyethyleneglycol (PEG) can prevent the formation of aggregates [260], yet brings along other problems. Concerns have been raised that PEGylation of lipoplexes may be unfavorable in the context of vaccination as this could inhibit uptake of nanoparticles by APCs and negatively interfere with the release of complexes from the endosomal compartment [213]. However, a study performed by Singh and colleagues demonstrated that subcutaneous immunization of rabbits with PEGylated lipoplexes carrying a synthetic gp41 epitope of HIV-1 induced two times higher immune responses and prolonged persistence of antibodies than liposomes

Conceptually, particulate vaccines mimic the particulate nature of pathogens, including the size (nano- to micro-meter range), which facilitates their uptake by APCs. The actual size of the particles influences the uptake mechanism by APCs and the way of antigen presentation. Generally, larger particles are predominantly internalised via phagocytosis or macropinocytosis, while smaller particles are taken up by other endocytic mechanisms [262- 264]. Internalisation through phagocytosis is known to lead to antigen cross-presentation in DCs [265, 266]. This emphasizes the enhanced potential of particulate antigen delivery to induce cellular immune responses as compared to soluble antigens, which are poorly cross-

Most nano- or microparticles can be functionalized with ligands or antibodies directed against cell surface receptors to target specific tissues or cells [267]. DCs express lectin-like receptors such as mannose receptor, DEC-205 and DC-SIGN which are believed to be involved in the phagocytosis of pathogens [268]. One way of increasing or promoting particle uptake by DCs is the attachment of mannose groups recognized by the mannose

presented and preferentially presented via the MHC class II pathway.

In summary, targeting of particles may not only enhance efficacy but also the specificity of interaction with the surface receptors on DCs [179]. Moreover, coupling of specific adjuvants (e.g. TLR ligands), with the aim to promote the desired immune response, can also positively influence the uptake by DCs [280].

#### **6.5. Co-delivery of antigens and adjuvants to improve immunogenicity of DCs**

Although particulate antigen delivery improves antigen uptake and presentation by DCs, most particles are not immunogenic by themselves. This offers a possibility to use a specific adjuvant that can skew towards the desired type of immune responses [187]. Despite some positive results in animal models, one should be aware of the toxicity of potential immunomodulators in humans, that jeopardizes their clinical use [281]. Aluminum salts (referred to as alum) are the oldest and most widely used adjuvants for human vaccines. It has been shown that antigens can be precipitated with alum to form colloid particles, creating a depot effect after vaccination, that elicits strong humoral immune responses [282]. These results, together with further observations clearly showed that the physical linkage of antigen and immunomodulator is crucial to induce strong immune responses [182, 183, 283].

Immunostimulating complexes (ISCOMs) are another interesting delievery system. It is a sort of liposomal delivery vehicle with a built-in adjuvant. They are composed of a protein antigen, phosopholipids and the saponin Quil A adjuvant, derived from the bark of the *Quillaja saponaria*, a South American tree. It has been demonstrated that the use of ISCOMs improves CTL responses of influenza virus based vaccines [284, 285]. ISCOM-based vaccines have been tested in animal models and in clinical trials against cancer [286] and viral infections [287-289] [290, 291]. In all studies satisfactory safety and tolerance were shown and both humoral and cellular responses were induced.

Several studies have reported the use of ISCOMs as a system to deliver HIV or SIV antigens. In non-human primate models, incorporation of HIV or SIV peptides into ISCOMs has been shown to induce protective immunity [288, 292, 293]. Moreover, studies in mice demonstrated that ISCOMs can be used to elicit immune responses against HIV-1 antigens [287]. To generate mucosal immunity, Koopman and colleagues immunized rhesus

maquaqes intranasally or via lymph nodes with HIV-1 peptides formulated into PR8-Flu ISCOMs [294]. Intranodal injection of these ISCOMs induced strong systemic and mucosal immune responses. In contrast, intranasal application resulted in very weak responses. Currently, ISCOM-based vaccines have been approved for veterinary use and are undergoing clinical trials for human use [295].

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 53

The extracellular TLRs mainly recognize bacterial invaders, but also fungi and some enveloped viruses. Bacterial lipoproteins are recognized by the heterodimers TLR1:2 (triacyl lipoproteins) and TLR2:6 (diacyl lipoproteins). Liposomes engrafted with palmitoyl chain lipopeptides have been shown to up-regulate DC maturation markers *in vitro* [298]. Lipopolysaccharide (LPS), a gram negative bacterial carbohydrate, is recognized by TLR4. Recently, a phase I clinical trial has started evaluating the potential of autologous dendritic cells, *ex vivo* loaded with lipopeptides and activated with LPS, as an immunotherapy in HIV infected patients (NCT00796770). Lipopolysaccharide (LPS), a gram negative bacterial carbohydrate, is recognized by TLR4. Since the *in vivo* use of LPS for human purposes is not possible due to the risk of septic shocks, a derivative, monophosphoryl lipid A from *Salmonella Minnesota* referred to as MPLA, was proposed as a substitute. It has been shown to be less toxic than LPS and thus preferable for human vaccine applications. MPLA has been incorporated in PLGA particles and liposomes [299, 300]. Furthermore, MPLA is already used in combination with alum (AS04) as an adjuvant in Cervarix®, a human papilloma virus vaccine from GlaxoSmithKline. Another bacterial component, flagellin, is recognized by TLR5 and is also used as a potential adjuvant. Flagellin-related peptides containing a His-tag and incorporated in liposomes containing a tumor antigen, induced antitumor responses resulting in complete tumor regression in mice [278]. The advantage of flagellin is that it can be co-integrated as a protein into particulate vehicles or integrated as

The intracellular TLRs specifically recognize nucleic acids and are of major importance to the recognition of double stranded DNA (dsDNA) from viral or bacterial intruders. TLR-9, only expressed in pDCs, recognizes unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs). CpG motifs were demonstrated to induce cell-mediated responses *in vivo* when incorporated into PLGA particles [301, 302], liposomes [303] and other microparticles [304]. All these studies reported enhanced Th1 biased immune responses. Viral single stranded RNA (ssRNA) is recognized by TLR7 (expressed by all DC subsets and B-cells) and TLR8 (expressed by macrophages, monocytes and myeloid DCs) [181]. Agonists used to trigger these receptors are the synthetic imidazoquinoline drug compounds imiquimod and resiquimod (R848) or polyuridylic acid (polyU). Only few studies so far have used TLR7/8 ligands for particulate antigen delivery. Johnston *et al.* demonstrated that the combination of TLR 7 ligand with liposomes increased the cellular immune responses against OVA protein in a mice model [305]. Another study reported that co-encapsulation of OVA antigen and polyU in PLA-DOTAP microparticles increased the antibody titers and numbers of IFN-γ secreting T cells [306]. Finally, the endosomal TLR3 recognizes dsRNA, which can be mimicked by the synthetic polyinosinic:polycytidylic acid (poly I:C). This dsRNA analog is widely evaluated as a potential adjuvant since it strongly promotes the maturation of DCs [184, 204]. Furthermore, the combination of poly I:C with protein antigens encapsulated in either polymeric [183, 184, 307] or liposomal [308] particles favors enhanced Th1 immune responses. With regard to mRNA vaccination, the use of endosomal TLR ligands is problematic, since upon recognition by their receptor, high levels of type I interferon are produced leading to the activation of RNases [309, 310]. However, mRNA by itself can also trigger endosomal TLRs, implying the crucial importance of

stabilizing the antigen encoding mRNA (reviewed by Tavernier *et al.*) [189].

His-tagged peptides into liposomes.

As described before, for an optimal immune response it is crucial that DCs, besides actively taking up the antigen, undergo activation and maturation to stimulate effective T cell responses. Ligands mimicking PAMPs that can target PPRs are therefore of potential interest. Extracellular and intracellular PPRs are divided into four groups: TLRs, NLRs, CLRs and RLRs [181]. Depending on the receptor that is triggered, DCs produce and secrete various sets of cytokines, determining the type of immune response [296]. Moreover, the incorporation of TLR ligands promotes phagocytosis of APCs [280]. TLRs represent the majority of PPRs studied in the development of effective adjuvants [281]. TLRs can be divided into two groups: the surface bound receptors (TLR 1, 2, 4, 5, 6) and the intracellular receptors present in the endosomes (TLR 3, 7, 8, 9) (**table 4**).


**Table 4.** Overview of Toll like receptors (TLRs) with their ligands and effect of activation [297].

The extracellular TLRs mainly recognize bacterial invaders, but also fungi and some enveloped viruses. Bacterial lipoproteins are recognized by the heterodimers TLR1:2 (triacyl lipoproteins) and TLR2:6 (diacyl lipoproteins). Liposomes engrafted with palmitoyl chain lipopeptides have been shown to up-regulate DC maturation markers *in vitro* [298]. Lipopolysaccharide (LPS), a gram negative bacterial carbohydrate, is recognized by TLR4. Recently, a phase I clinical trial has started evaluating the potential of autologous dendritic cells, *ex vivo* loaded with lipopeptides and activated with LPS, as an immunotherapy in HIV infected patients (NCT00796770). Lipopolysaccharide (LPS), a gram negative bacterial carbohydrate, is recognized by TLR4. Since the *in vivo* use of LPS for human purposes is not possible due to the risk of septic shocks, a derivative, monophosphoryl lipid A from *Salmonella Minnesota* referred to as MPLA, was proposed as a substitute. It has been shown to be less toxic than LPS and thus preferable for human vaccine applications. MPLA has been incorporated in PLGA particles and liposomes [299, 300]. Furthermore, MPLA is already used in combination with alum (AS04) as an adjuvant in Cervarix®, a human papilloma virus vaccine from GlaxoSmithKline. Another bacterial component, flagellin, is recognized by TLR5 and is also used as a potential adjuvant. Flagellin-related peptides containing a His-tag and incorporated in liposomes containing a tumor antigen, induced antitumor responses resulting in complete tumor regression in mice [278]. The advantage of flagellin is that it can be co-integrated as a protein into particulate vehicles or integrated as His-tagged peptides into liposomes.

52 Immunodeficiency

undergoing clinical trials for human use [295].

1:2 Triacyl lipoproteins Mo-DCs,

2 Lipoproteins Mo-DCs,

4 Lipopolysaccharide Mo-DCs,

5 Flagellin Mo-DCs,

6:2 Triacyl lipoproteins Mo-DCs,

3 Double stranded RNA

7 Single stranded mRNA

8 Single stranded mRNA

9 Double stranded DNA

receptors present in the endosomes (TLR 3, 7, 8, 9) (**table 4**).

**TLR TLR ligand DC subset Effect of activation**

myeloid DCs

myeloid DCs

myeloid DCs

myeloid DCs

myeloid DCs

pDC, myeloid

DCs

Mo-DCs, myeloid DCs

maquaqes intranasally or via lymph nodes with HIV-1 peptides formulated into PR8-Flu ISCOMs [294]. Intranodal injection of these ISCOMs induced strong systemic and mucosal immune responses. In contrast, intranasal application resulted in very weak responses. Currently, ISCOM-based vaccines have been approved for veterinary use and are

As described before, for an optimal immune response it is crucial that DCs, besides actively taking up the antigen, undergo activation and maturation to stimulate effective T cell responses. Ligands mimicking PAMPs that can target PPRs are therefore of potential interest. Extracellular and intracellular PPRs are divided into four groups: TLRs, NLRs, CLRs and RLRs [181]. Depending on the receptor that is triggered, DCs produce and secrete various sets of cytokines, determining the type of immune response [296]. Moreover, the incorporation of TLR ligands promotes phagocytosis of APCs [280]. TLRs represent the majority of PPRs studied in the development of effective adjuvants [281]. TLRs can be divided into two groups: the surface bound receptors (TLR 1, 2, 4, 5, 6) and the intracellular

Upregulation of CCR7, IL-6, IL-10, IL-

Upregulation of CCR7, IL-6, IL-10, IL-

IFN-α/β activation and up-regulation IL-

Upregulation of CD80, CD86, CD83, CCR7. Secretion of IL-6, IL-8, IL-10, IL-12p70, IFN-

Upregulation of CD80, CD86, CD83, CCR7. Secretion IFN-α/β , IL-1 , TNF-α, IL-8, IL-

Upregulation of CCR7, IL-6, IL-10, IL-

Upregulation of CCR7,CD40, CD80 and CD86. Secretion of IL-12p70 (myeloid DCs).

HLA-DR, CCR7. Upregulation of IFN-α (very high), IFN-β (lower), IL-6, TNF-α

Secretion of IFN-α (pDCs).

pDC Upregulation of CD40, CD80, CD86, CD83,

Mo-DCs Increased TNF , IL-8, IL-12p40, MCP-1, CCL2, CCL3, CCL4, CCL5

12p70,TNF-α

12p70,TNF-α

12p70

12p40

12p70,TNF-α

(low), IL-8

**Table 4.** Overview of Toll like receptors (TLRs) with their ligands and effect of activation [297].

β

The intracellular TLRs specifically recognize nucleic acids and are of major importance to the recognition of double stranded DNA (dsDNA) from viral or bacterial intruders. TLR-9, only expressed in pDCs, recognizes unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs). CpG motifs were demonstrated to induce cell-mediated responses *in vivo* when incorporated into PLGA particles [301, 302], liposomes [303] and other microparticles [304]. All these studies reported enhanced Th1 biased immune responses. Viral single stranded RNA (ssRNA) is recognized by TLR7 (expressed by all DC subsets and B-cells) and TLR8 (expressed by macrophages, monocytes and myeloid DCs) [181]. Agonists used to trigger these receptors are the synthetic imidazoquinoline drug compounds imiquimod and resiquimod (R848) or polyuridylic acid (polyU). Only few studies so far have used TLR7/8 ligands for particulate antigen delivery. Johnston *et al.* demonstrated that the combination of TLR 7 ligand with liposomes increased the cellular immune responses against OVA protein in a mice model [305]. Another study reported that co-encapsulation of OVA antigen and polyU in PLA-DOTAP microparticles increased the antibody titers and numbers of IFN-γ secreting T cells [306]. Finally, the endosomal TLR3 recognizes dsRNA, which can be mimicked by the synthetic polyinosinic:polycytidylic acid (poly I:C). This dsRNA analog is widely evaluated as a potential adjuvant since it strongly promotes the maturation of DCs [184, 204]. Furthermore, the combination of poly I:C with protein antigens encapsulated in either polymeric [183, 184, 307] or liposomal [308] particles favors enhanced Th1 immune responses. With regard to mRNA vaccination, the use of endosomal TLR ligands is problematic, since upon recognition by their receptor, high levels of type I interferon are produced leading to the activation of RNases [309, 310]. However, mRNA by itself can also trigger endosomal TLRs, implying the crucial importance of stabilizing the antigen encoding mRNA (reviewed by Tavernier *et al.*) [189].

Another possibility to improve immunogenicity is the addition of co-stimulatory molecules. The addition of CD40L to DCs loaded with liposome-complexed HIV-1 proteins could prime HIV-1 specific CD8+ T cells *in vitro* [162]. Within the scope of mRNA transfection, mRNA encoding co-stimulatory molecules can be co-transfected with mRNA encoding an antigen, which has been shown to strongly enhance the capacity of electroporated DCs to stimulate HIV-specific T cell responses [311].

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 55

This work was supported by grants from IUAP (Inter-University Attraction Poles, P6/41 of the Belgian government), FWO (Fund for Scientific Research Flanders, project number G.0226.10) and SOFI-B (Secondary Research Funding ITM). W. De Haes is a doctoral fellow of the Institute for Science and Technology (IWT), Flanders. There is no conflict of interest.

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**Acknowledgement** 

**8. References** 
