HIV Vaccines: A Brief Overview

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1 REVIEW doi: /sji HIV Vaccines: A Brief Overview D. Lema, A. Garcia & J. B. De Sanctis Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela Received 24 February 2014; Accepted in revised form 22 April 2014 Abstract The scope of the article is to review the different approaches that have been used for HIV vaccines. The review is based on articles retrieved by PubMed and clinical trials from 1990 up to date. The article discusses virus complexity, protective and non-protective immune responses against the virus, and the most important approaches for HIV vaccine development. Correspondence to: Dr J. B. De Sanctis, Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Ciudad Universitaria, Caracas 1050, Venezuela. s: sanctisj@gmail.com, juan.desanctis@ucv.ve Introduction More people today have access to life-saving antiretroviral therapy for HIV/AIDS than ever before. Yet for every person that starts pharmacological treatment, three others are infected. Treatment alone will not curtail the HIV/ AIDS pandemic. To control the pandemia, powerful tools for disease prevention have to be widely accessible. Vaccines have been the most effective, safe and economic approach to prevent and eradicate infectious diseases. Developing a safe and effective HIV vaccine has been a difficult task because HIV has proven to be a tough target. The results of clinical trials have lead to new strategies [1]. Immune responses that correlate with HIV-1 protection A correlate of risk is a measured immune variable that is associated, statistically or mechanistically, with infection rate, disease protection and vaccine efficacy [1, 2]. Due to the lack of natural protection against HIV infection, this number is basically unknown, and it may only be calculated after successful vaccine development [3]. Up to date, immune response elicited in non-human primates (NHP) experiments or Phase I/II human trials, have not been successful [4 6] maybe because natural resistance to HIV is likely to be a multifactorial process involving host genetics, innate and adaptive immune responses, and virus diversity. The presence of individuals who are highly exposed to HIV-1 but do not get infected and long-term nonprogressors, provide hope for a better understanding of correlates for protection that may lead to a more effective vaccine strategy. Highly exposed seronegative (HESN) populations have been identified among intravenous drug users, children born to seropositive mothers, discordant couples and commercial sex workers. The findings in 1996 on the protective effect of the CCR5 delta 32 deletion allele in HIV-1 infection shifted focus to host genetics as probable cause of HIV-1 resistance [7 9]. Humoral immunity Broad neutralizing antibodies Studies of neutralizing antibodies in HIV-1 infected individuals provide insights into the quality of the response that should be possible to elicit with vaccines and ways to design effective immunogens. Some individuals make high titres of exceptional broadly reactive neutralizing antibodies (bnabs) that are of particular interest; however, more modest responses may be a reasonable goal for vaccines. Viral epitopes in HIV natural infection are strain-specific, highly variable, highly mutagenic and rapidly evolve to escape immune recognition. These antigens induce the expansion of CD4 T follicular helper cells, which is associated with an increase of germinal centre B cells and plasma cells as well as IgG1 hypersecretion, leading to perturbations of B cell differentiation, resulting in dysregulated antibody production [10, 11]. After 3 years of infection, 20 30% of patients produce neutralizing antibodies capable of neutralizing heterologous HIVs, which target structurally conserved, vulnerable, Env epitopes. The titres of these antibodies seem to correlate with time of infection and viral plasma load. Broadly neutralizing antibodies effectiveness depends on maturation affinity, somatic mutations, polyreactivity and Ó 2014 John Wiley & Sons Ltd 1

2 2 HIV Vaccines D. Lema et al. structural characteristics in the CDR-H3 region of the immunoglobulin. Nevertheless, the high frequencies of mutations induce the assembly of a variety of nonneutralizing antibodies that, in turn, decrease bnab production [12 16]. Several antibodies that bind the CD4-binding region of gp120, gp140 and gp41 have been identified and it has been proposed that these antibodies neutralize the virus by blocking cell receptors, or by inactivating the virus. The most relevant reports reveal that: (1) VCR01 antibody targets a gp120 invariable region, independent of CD4 binding, a feature shared with b12; (2) attempts to elicit antibodies similar to the natural counterpart b12 have proven unsuccessful; (3) the quaternary antibodies PG9 and PG16 (rarely present natural infection), target conformational conserved discontinuous epitopes of the V1/V2 loops of gp120, and are able to neutralize 80% of circulating HIV-1 isolates; (4) the HGN194 antibody recognizes the V3 loop and neutralizes a range neutralization-sensitive and resistant viruses; (5) the 2G12 antibody binds to the oligomannose cluster residue of gp120 and recognizes three possible epitope binding sites; 5) the X5 antibody binds to the CCR5 co-receptor binding site of gp120, only transiently exposed after CD4 binding by gp160 [17 20]. Another important target for bnabs is the membraneproximal external region (MPER) of gp41, which contains structurally conserved epitopes exposed after engagement of viral spikes with their cell receptors. Antibody binding to MPER interferes with viral fusion to the cell and thus impedes infection. Antibodies 2F5, 4E10, 10E8 and Z13e1 target gp41 epitopes and have been demonstrated in 27% of healthy HIV-1 infected individuals [18 21]. Passive immunization with 2F5 or 4E10 offered protection against rectal simian human immunodeficiency virus (SHIV) [18]. Nonetheless, the epitope region has cardiolipin-like properties, and it may be tolerogenic [22]. On the other hand, the antibody 10E8 has been shown to neutralize 98% of tested viruses, it does not bind lipids and it is nonautoreactive [22]. Passive immunization with bnabs provided protection, even across clades, of rhesus macaques, in vivo and in vitro [18]. These results suggest that inducing bnabs by vaccination should provide protection to humans; however, no current vaccine elicits this response. An alternative approach is to generate a vaccine able to prime germline B cells with a relevant HIV immunogen, produce bnabs and later boost with other immunogens to mimic virus diversification [14, 15, 27, 28]. The aim is to generate bnabs and elicit a protective immune response. The main difficulty is to find the right immunogens. Non-neutralizing antibodies Only about 1% of individuals, termed elite neutralizers, show unusually potent serum neutralization [8].Interestingly, elite controllers produce less neutralizing antibodies than other patients [23]. Thus, non-neutralizing antibodies may have an important role in infection control [23]. The protective effect of non-neutralizing antibodies is dependent upon FccR-mediated functions by recruiting antigen presenting cells (APCs), promoting antibody-dependent cell-mediated cytotoxicity (ADCC) and inhibiting viral replication via antibody-dependent cell-mediated virus inhibition (ADCVI) involving antiviral cytokine and protease secretion [24 26]. In HIV, NK cells kill infected cells, predominantly, by ADCC [24 26]. The common antigens for these antibodies are Env s gp120, Nef, Pol and Vpu proteins [26]. Viral inhibitory activity, ADCVI, was reported with b12, 2G12, 447-D and most MPER-specific antibodies [18 21]. Thus, bnabs possess dual (neutralizing and non-neutralizing) protective activity. Both ADCC and ADCVI activities improve after infection and are enhanced by antibody maturation. ADCVI appears as early as the cytotoxic T cell response, about 1 week after symptom onset or 1 month after HIV exposure [23 25]. ADCC is enhanced in elite controllers, not in disease progressors, suggesting that this mechanism is crucial for infection control [23]. These non-neutralizing antibodies, reported in HIV vaccine trials, contrast with neutralizing antibodies, as are easily generated, do not require B cell maturation and offer immune protection by ADCC and ADCVI [15 19, 26, 27]. Mucosal antibodies might offer protection in early viral infection [28]. They interact with specific receptors and also block the transcytosis used by the virus to penetrate the exocervical, rectal or intestinal epithelium [29]. Their potential in vaccine development is underlined by the fact that, in macaques, vaginal application of neutralizing and non-neutralizing antibodies prevented infection and reduced viral load, respectively, after a SHIV challenge [29]. A conserved gp41 epitope, QARVLAVERY, induced mucosal IgA production, neutralized the virus and inhibited transcytosis [30]. Even though non-neutralizing antibodies do not inhibit CD4+ T cell infection, as neutralizing antibodies do, they obstruct APC infection, target of HIV in mucosal surfaces during sexual HIV transmission, thus are useful blocking viral infection [23, 25]. Cellular immunity It is thought that cellular immunity is more important than humoral immunity in the control of HIV infection. Some HIV controllers show a marked cellular immune response, probably due to a combination of low amounts of regulatory T cells and a high-vigorous T cell response [31]. HIV viral control is associated with a higher frequency of HIV-specific IL-2 secreting immature CD4+ T cells and HIV-specific mature CD8+ T cells, and the former probably maintains the latter population [31, 32]. In these subsets, CD4+ expressing eomesodermin transcription Scandinavian Journal of Immunology, 2014, 80, 1 11

3 D. Lema et al. HIV Vaccines 3 factor can kill infected cells, probably through perforin and granzyme A and B secretion [33]. Granzyme A secretion by these cells correlates with a slower disease progression [33], and it is hypothesized that this CD4+ cytolytic activity is dependent on antigen presentation through MHC-II, which facilitates killing of infected macrophages [33, 34]. Yet, the role of cytolytic CD4+ T cells in HIV infection remains unclear. Understanding exactly how CD4+ T cells coordinate the immune system, including directing the quality and persistence of protective CD8+ T cell and B cell responses, is likely to be crucial for increasing the effectiveness of candidate vaccines. The phenotypes of CD8+ T cells that correlate with lower viral loads in chronic HIV infection are either central or effector memory cells that do not express exhaustion markers such as PD-1 [35]. These are polyfunctional cells capable of cytokine secretion, proliferation and cell lysis [35]. Certain HLA alleles, along with several SNPs in the HLA-B peptide groove, are associated with rapid or slow disease progression [34, 35] and are responsible for 19% of host control [34 36]. Cytotoxic T lymphocyte (CTL) activity vary depending on the infecting HIV isolate and clade [34 36]. CTL activity against Gag p17 and p24 proteins is important; both contain immunodominant and subdominant epitopes [36, 37]. The relative dominance of epitopes is determined by their abundance in cell surface (epitope presentation), which is in turn determined by epitope processing, including proteasome digestion, TAP processing, ERAAP trimming and HLA binding [37]. CTL responses to the p24 immunodominant epitope KRWIILGLNK are associated with low viral titres and slow disease progression, while CTL targeting p17 epitopes SLYNTVATL and RSLYNTVATLY, commonly immunodominant in chronic infection, are not associated with slow disease progression [37]. Both intra-epitopic as well as extra-epitopic mutations (polymorphisms in aminoacid residues neighbouring conserved epitopes) are known to affect epitope processing and presentation [36, 37]. The high-rate mutations of infecting HIV-1 virus generate new epitopes, affecting the amount of known immunodominant CTL epitopes and CTL effectiveness. Thus, an epitope-based vaccine should be designed considering immunodominant epitopes, their flanking regions and sequences to enhance antigen processing and presentation for an optimal activation of T cells. Vaccines An effective HIV vaccine should induce powerful and durable immunity such as to prevent infection in healthy individuals and/or reduce viral replication and viral load in infected ones, slowing or halting disease transmission and progression. The first experimental immunization of human against the AIDS retrovirus HIV-1 was started in a series of HIV seronegative healthy volunteers in November 1986, using vaccinia virus recombinant (V25) expressing gp160 env determinants of HTLV III B at the surface of infected cells, four different protocols were used, for the first time the results show that an immune stage against HIV can be obtained in a man [38]. Since then, more than 256 trials (concluded or ongoing mostly Phase I or II), involving over 44,000 healthy human volunteers have tested HIV vaccine candidates [38 43]. Of these trials, only six (VAX004, VAX003, Step, Phambili, RV114 and HVTN 505) have reached clinical efficacy (Phase IIb and III) (Table 1). The first advances intended to induce neutralizing antibodies; however, difficulties were encountered with the immunogen. The focus of HIV vaccines then turned on to CD8+ T cell responses, which seemed to be a logical Table 1 The table illustrates the different documented HIV vaccine trials, the type of vaccine, the desired immune response and the result of the trial. HIV vaccine efficacy trials Trial Date Phase Vaccine type Desired immune response Result VAX III rgp120 (clade B) Humoral No efficacy VAX III rgp120 (clades B + E) Humoral No efficacy step (ceased for possible increase in HIV acquisition) IIb rad5 (gag/pol/nef) Cellular Increased HIV infection risk in MSM and uncircumcised men Phambili (stopped due to step IIb rad5 (gag/pol/nef) Cellular No efficacy results) RV III canarypox (gag/pol/nef) + rgp120 Cellular + humoral 31.2% efficacy HVTN (prematurely stopped in 2013 due to initial results showing that the vaccine was ineffective in preventing HIV infections and lowering viral load among those participants who had become infected with HIV) IIb DNA plasmid (gag/pol/nef/env) + rad5 (gag/pol/env) Cellular + humoral No efficacy Ó 2014 John Wiley & Sons Ltd

4 4 HIV Vaccines D. Lema et al. approach [38 43]. The scope was to induce a long-lived memory CD8+ T cell population, capable of rapid killing infected cells in secondary exposures to the virus. Instead, the results of the step trials were disappointing and generated new doubts on the strategy used. Hence, a combined approach, bnabs neutralizing infecting viruses along with an efficient cellular response (capable of eliminating infected cells that bypassed humoral immunity), seems desirable but is difficult to achieve just by simple vaccination [43]. Many vaccine schemes have been attempted against HIV-1, and every step in their design from antigen, delivery system, adjuvant and booster strategy have been carefully analyzed. Live attenuated vaccines, inactivated virus vaccines, virus-like particles, subunit vaccines and DNA vaccines have been developed and tested. As a result, different immune responses have been elicited, which have been difficult to analyze. Several experiences have promoted new discoveries as follows: (1) new adjuvants were developed [44 46], (2) booster regimens were redesigned to enhance effector memory cell intended for peripheral tissues (homing for lymph nodes to control HIV replication) [47, 48] and to decrease refractory memory CD8+ T cells [48], (3) therapeutic vaccines, peptides plus dendritic cells (DC), were developed to enhance HIV immunity to eliminate the virus completely even after antiretroviral therapy discontinuation [49]. However, the latent infected cell population, which would need to be eliminated by a robust immune cell vaccine-elicited response, might not be susceptible to cytolytic CD8+ T cell killing, and consequently, it may evade immune system control [50, 51]. As virus variability is a key important issue, two different approaches were proposed as follows: (1) to target constant epitopes, using constant immunogens and (2) to use mosaic antigens to broaden immune response against the virus [52, 53]. Engineered synthetic genes that only express the consensus, conserved, HIV envelope epitopes, elicited antibodies to a wide span of HIV pseudoviruses as compared to using wild-type Env proteins [54]. In rhesus macaques, mosaic antigen vaccines expanded the span (diversity of recognized epitopes) and depth (diversity of recognized variants for a given epitope) of the specific immune response in CD8+ and CD4+ cells without affecting the neutralizing antibody response [53]. It is has been shown to be an interesting tactic to improve vaccine efficacy. Many strategies have been undertaken to enhance the immunogenicity of native HIV molecules [55 59]. The gp140 trimer (gp160 with gp41 transmembrane and intracytoplasmatic domains deleted) is a better immunogen than single peptides [54]. Crystallographic studies of the complex envelope proteins-antibody have generated relevant information for structure-based epitopes [22, 55, 56] including virus evolution experiments. In rabbits, an enhanced antibody production to neo-epitopes and neutralization of certain HIV strains was recorded [55]. This result suggests that efforts should be focusing on the HIV-specific immune response rather than only an antigenspecific vaccine. Another interesting proposal was to design an immunogen with a conformational gp120 or gp140 epitope bound to poorly immunogenic CD4 molecules to avoid autoantibody production. Rabbits immunized with a covalent CD4-gp120 or CD4-V2-deleted gp140 complex produced high titres of CD4-induced epitope-specific neutralizing antibodies; however, the titres were not significantly higher as compared to those achieved with native Env alone [57]. Live attenuated and inactivated virus vaccines Whole virus vaccines are processed and presented via MHC-I inducing a cellular response in a manner resembling natural infection. Both live attenuated and inactivated virus vaccines have proven to offer immunity in NHPs. Formalin inactivated simian immunodeficiency virus (SIV) induced protection in the majority of vaccinated macaques against intravenous pathogenic virus [58]. Some attenuated nef-deleted SIV vaccines have shown success in inducing complete immunity in macaques [59]. Macaques immunized with a nef and vpr-deleted SIV strain, SIVmac239D3, were protected against disease and reduced viral load when challenged intravenously with highly pathogenic SIV and SHIV strains [60]. However, immune response is not constantly elicited [60]. Although animal studies with whole virus vaccines show progress, viruses have not been tested in humans owing to safety concerns [61]. As viruses stimulate a vigorous immune response, low-grade persistent infection with continuous antigenic exposure, more efforts have been directed to produce replication competent viral vectors for vaccine delivery [61, 62]. Hence, several replication competent viral vector vaccines have been developed for animal testing and human trials, including adenovirus, poxvirus, yellow fever virus, Venezuelan equine encephalitis virus and other promising immunogens [22, 40]. Protein subunit vaccines In the light of the findings that an adequate neutralizing antibody response could offer protection in NHPs, early vaccine developments were designed with this goal [22, 40]. It seemed logical to use Env, the only HIV neutralizing antibody target site, with adequate adjuvants to elicit a protective humoral immune response. Vaccines from HIV-1 protein subunits gp160, gp120 and gp140 have been developed, including the first HIV vaccine to reach Phase I trial, a recombinant gp160 molecule produced in a baculovirus insect cell system [39]. They produce neutralizing antibodies and activate CD4+ T cells; however, they do not produce a CD8+ CTL response [43], which is an important drawback. Scandinavian Journal of Immunology, 2014, 80, 1 11

5 D. Lema et al. HIV Vaccines 5 In the 1990s, the AIDSVAX B/B vaccine (VaxGen), employing HIV-1 clade B recombinant gp120 envelope proteins, and a primary clade B isolate (GNE8), with alum salts as adjuvant, showed good safety and immunogenicity in Phase I and II trials [61 63]. In 1998, the VAX004 trial became the first Phase III HIV vaccine candidate, testing the vaccine in North America and the Netherlands [64]. A similar vaccine, AIDSVAX B/E, MN clade B gp120 with a bivalent clade E rgp120 protein from a primary isolate (A244) as the second antigen, passed Phase I/II trials in Thailand [64, 65]. Both trials recruited volunteers in population in high risk for HIV infection, VAX004 enrolling 5,417 men that have sex with men (MSM) and women at high heterosexual infection risk and VAX intravenous drug users (IDUs) [65]. Neither vaccine offered protection to infection, reduced viral load, nor slowed disease progression despite the presence, in some individuals, of neutralizing and non-neutralizing Envspecific antibodies [64 66]. ADCVI-mediating antibodies did inversely correlate with HIV infection probability; on the other hand, neutralizing antibodies did not [66]. Thus, an effective vaccine would require mucosal antibody response along with a robust cellular immunity against the virus. The interest in protein-based vaccines has been lost due to poor immunogenicity. New strategies have been developed to overcome this drawback. DC targeting has been shown to be a useful strategy. Homing the antigen to DC cells with anti-dec205 antibody potentiated both humoral and CD4+, but not CD8+, Gag-specific immune responses in mice [67] Viral-like particles Viral-like particles (VLPs) are structures, manufactured in vitro, combine a non-replicating, non-pathogenic viral vector with the desired antigen. They retain the vector s cell binding ability, which results in antigen presentation via MHC class I and II [68]. Recent attempts with VLPbased vaccines have been largely disappointing. Pastori et al. [69] created a (AP205) bacteriophage VLP expressing 6 HIV-1 gp41 alpha-helix regions. Administration in mice via subcutaneous or intranasal routes elicited high-specific antibody titres, including ADCC antibodies with a reduced span of neutralizing antibodies. AVX101, a VEEV replicon-based vaccine, with promising results in preclinical tests, showed modest immunogenicity in Phase I trials [70]. Again, more research is needed to address the lack of response of these vaccines. Live recombinant vaccines A method of gene delivery to host tissues is the use of a viral vector containing the desired genes to be introduced. When these recombinant viruses enter host cells, with the foreign gene, they induce antigen expression (MHC class I and II), phagocytosis of apoptotic antigen-expressing cells and induction of CD4+ T cell responses [71]. The most used viral vectors are poxviruses, including canarypox, fowlpox, but modified vaccinia virus ankara and the modified Copenhagen strain NYVAC [71 73]. These viruses, along with Ad26 and Ad35, predominate in clinical trials [73] as they are naturally or artificially replication-deficient in primates, but also less immunogenic than other structures. Given the fact that some of these vectors suppress immune responses, immunogenicity was enhanced by the deletion of interferon pathwayblocking genes in MVA or NYVAC [72]. Also, preexisting immunity to some vectors might negatively affect vaccine efficacy [74] while it does not affect others, fowlpox and canarypox, [71]. Thus, the use of distinct, more effective, vector serotypes, such as Ad26 and Ad35, might enhance future developments in this field [74, 75]. The failure of the VAX003 and VAX004 trials, which elicited a B cell response, brought attention to T cell vaccines as an alternative to HIV protection. The step study (HVTN 502) was the first completed trial to evaluate whether a vaccine inducing cellular immunity to HIV-1 could protect against infection or reduce viral load [76 78]. The vaccine tested, developed by Merck Research Laboratories, was a recombinant live vector composed of replication-defective recombinant adenovirus 5 (rad5) expressing HIV-1 clade B gag, pol and nef [76 78]. As env was not included, a neutralizing antibody response was not to be elicited. Cellular immunity was the scope of the vaccine. The study enrolled high-risk populations, mainly MSM, but also heterosexual males and females in North and South America, Australia and the Caribbean. The Phambili sister trial (HVTN 503) tested the same vaccine regimen in South African heterosexual males and females [76, 79]. Both studies were terminated early due to higher infection rates in the vaccinated group, despite finding, for the first time, IFN-c secreting HIV-specific T cells by ELISPOT assays [79, 80]. HIV-specific CD4+ and CD8+ T cells were identified in 41% and 73% of the individuals (32% for both) [76]. However, cell activations do not differentiate between HIV cases and non-cases and/or cytokine profiles [76]. Vaccination did not affect the course of infection at a 2-year follow-up; time to initiate antiretroviral therapy, HIV RNA levels, CD4+ T cell counts, and AIDS-free survival [76]. Thus, IFN-c response was not associated with protection or lower infection rates. Post hoc analysis showed that the higher infection rate in the step trials was restricted to MSM who were uncircumcised and/or had high basal neutralizing antibody titres to Ad5 (2.3-fold increase in the latter) [77, 78]. Two hypotheses might explain this phenomenon as follows: (1) rad5 administration expands specific memory cell population, thus providing HIV more cellular targets to infect; Ó 2014 John Wiley & Sons Ltd

6 6 HIV Vaccines D. Lema et al. (2) the rad5 vaccine forms immune complexes with Ad5 neutralizing antibodies, promoting CD4+ T cell infection by HIV. The first hypothesis is largely refuted by the fact that basal Ad5 neutralizing antibody titres do not correlate with prior CD4+ or CD8+ T cell immunity, thus to memory cell population expansion and circulating CCR5+ T cell levels were not greater between patients with high and low Ad5 antibody titres [79]. Nevertheless, rad5 vaccine administration did not result in different rad5- specific CD4+ T cell trafficking to mucosal sites between rhesus macaques with high and low basal anti-ad5 antibody titres [80]. The second hypothesis, on the other hand, was strengthened by the finding that rad5-antibody complexes activate Langerhans cells, more than serum or Ad5 alone and render CD4+ cells vulnerable to HIV infection [73, 74]. The higher infection susceptibility decreased over the 18- and 36-month follow-up until it equaled the placebo group [78 80]. The Phambili trial was interrupted with <10% of the vaccinated participants due to step results. Analysis showed no statistical difference in HIV acquisition risk or viral set point between placebo and vaccine groups [80]. The trial used the same vaccine from step protocol; a clade B vaccine in a clade C area. The trial was justified on the basis of cross-clade CTL responses [79, 80]. These results have led to propose a combined vaccine: to prime with a live vector vaccine and boost with a protein subunit [79]. This strategy induces both a cellular and humoral responses and has yielded some of the most effective results to date, including a Phase III trial RV144. The Phase III HIV-1 vaccine trial RV144 was a test-ofconcept trial conducted by the Thai Ministry of Public Health and sponsored by the US Army Surgeon General, managed by the US Military HIV Research Program (MHRP) [81,82]. The vaccine tested was based on non-replicating recombinant canarypox virus (ALVAC) encoding clade B gag/pol and CRF01_AE env antigens (ALVAC-HIV vcp1521 from SanofiPasteur). It was administered in 2 primer doses, followed by 2 booster immunizations with the same ALVAC recombinant along with subtype B and CRF_AE rgp120s, (the VaxGen AIDSVAX immunogen) [83]. Clades B and E were chosen for the vaccine due to their local prevalence. Alum, instead of MF59, was used. The rationale was that using a live vector vaccine followed by a protein boost would induce both cellular and humoral immunity. The trial was launched in Thailand in 2003 [82, 83], it ended in 2009 and involved 16,402 volunteers (the largest vaccine trial to date) at risk of infection. In a modified intention-to-treat analysis (excluding patients infected at the first visit), the vaccine showed 31.2% efficacy (95% CI, ; P = 0.04) at preventing HIV infection [84], which is encouraging for future HIV vaccine development. The protection rate was higher among low and middle infection risk populations, compared with individuals with high infection risk [82]. The vaccine did not, however, reduced the viral load or CD4+ count on individuals who were subsequently infected after vaccination [82]. The most common response to the vaccine was a robust CD4+ T cell response to Env antigens [82, 83]. Only 24% of the individuals showed a CD8+ T cell response, <20% of the group showed an IFN-c ELISPOT response, and, in general, low titres of neutralizing antibodies were recorded. After Env priming, CD4+ intracellular cytokines were significantly higher in the vaccine as compared to the placebo group (34 versus 3.6%; P < 0.001) [84]. Serum virion capture antibodies were elicited in a fraction of individuals, including antibodies able to cross-react with transmitter/founder strains [85]. As with other rgp120-based vaccines, most volunteers developed ADCC activity [86]. It is unknown, however, if these antibodies are present in the mucosa. In summary, the trial showed a protection efficacy of 61% 1 year after vaccination, but it decreased to about half at the end of the trial. Given these results, changes in vaccination scheme regimen and adjuvant are underway [2, 39]. It has been reported that a high anti-env V1/V2 loop-specific and nonneutralizing, but ADCC enhancing antibody response was associated with a 71% decreased infection risk comparing low to medium responders; however, high anti-env IgA titres increased in the infection risk group [2, 87, 88]. These high specific IgA titres, possibly monomeric IgA, could compete with protective IgG [88]. DNA vaccines DNA vaccines mechanism of action relies on the administration of non-living, non-replicating, non-transmissible plasmids, which that are taken up by host cells (but very rarely integrated into their DNA). It encode proteins expressed by host tissues (predominantly skin and muscle), which induce class I and class II antigenic presentation. Cytoplasmic DNA sensing machinery may recognize the transfected plasmids and its metabolites and induce the production of type I IFNs [89]. However, these vaccines have been shown to induce low immunogenicity, due a low transfection efficiency, low DNA uptake by non-apcs and consequently, low antigen transcription rate. In vivo plasmid electroporation and cytokine coadministration have been effective increasing CD4+ and CD8+ responses [90]. One of the most difficult tasks is to enhance antigen presentation. Targeting DC surface molecules to induce cell homing and to facilitate antigen uptake has been attempted. A PD1-p24 DNA vaccine was able to enhance CD8+ (cytokine secretion, cytotoxicity), CD4+ T cell function and antibody titres in mice superiorly in comparison with an anti-dec205 approach [91] suggesting that PD-L1 homing antigens are effective vaccine enhancers. HIV DNA vaccines work better with primers in a heterogonous prime-boost or co-immunization strategy Scandinavian Journal of Immunology, 2014, 80, 1 11

7 D. Lema et al. HIV Vaccines 7 (administration of the same antigen in different vehicles) [93]. Mice immunized with a gag DNA, primed and boosted, recombinant Tiantan vaccinia virus were shown to exhibit potent Gag-specific T cell responses, independently of the doses used [92]. Likewise, DNA bound to mannosylated polyethyleneimine enhanced Gag-specific cellular and humoral responses in a murine DNA/Ad prime-boost protocol [92]. Co-immunizing mice and macaques with DNA and protein, rather than prime and booster schemes, provoked marked Env-specific humoral responses without jeopardizing cellular immunity [94]. In the latest clinical efficacy trial, HVTN 505 tested in circumcised MSM without pre-existing antivector antibody titres, a DNA prime-ad boost regimen was used [1]. The DNA prime consisted of 3 doses of a plasmid encoding env from HIV clades A, B and C, plus gag, pol and nef from clade B. The Ad5 boost encoded env from 3 clades and clade B gag and pol. The trial, which started in 2009, was scheduled to end in 2015, but was prematurely stopped in April 2013 due to lack efficacy [1, 95]. Lipoidal biocarriers Lipoid vesicular biocarrier systems for drug delivery have good bioavailability and pharmacokinetic properties [69, 96, 97]. These systems are suitable for encapsulation of both hydrophilic and hydrophobic drugs, enhance drug stability, increase therapeutic index, and delay elimination [69, 96, 97]. Certain products have been used in vaccination strategies as antigen delivery systems and adjuvants [69, 96, 97]. CAF01, a liposomal adjuvant system, is composed of cationic surfactant dimethyldioctadecylammonium and the immunomodulating glycolipid trehalose dibehenate, rendering a strong Th1 response and a humoral response [96, 97]. Charged cationic vesicles facilitate antigen adsorption at the site of administration, promoting APC presentation and stimulating strong Th1 and Th17 responses [97]. Moreover, virosomes induced mucosal antibodies protecting non-human primates against vaginal SHIV challenges [98]. A variety of liposomes have been studied for vaccine development against pathogens. Virosomes, vaccine candidates for HIV-1, influenza and hepatitis C virus vaccines, are reconstituted viral envelope lipid bilayers that function as antigen carriers and adjuvants. They are able to stimulate good antibody production and cellular immunity [96 98]. Virosomes may elicit either a CD4+ or CD8+ response as desired. Antigens attached to the virosome outer membrane will undergo APC endocytosis and thus MHC-II presentation. Antigens embedded inside the virosome are transported directly to the cytosol, and then processed, expressed via MHC-I, to elicit CD8+ responses. Thus both helper T cells and CTLs may be primed [99]. In contrast to other viral vector vaccines, pre-existing antibodies aid virosome delivery to APCs without inhibiting immune response [97]. Lipopeptides have been also used in vaccine strategies to activate DCs through TLR-2 [46, 97]. The LIPO-4 vaccine contains four palmitoyl-lysine associated Gag, Pol and Nef peptides covalently liked to tetanus toxin peptide (French National Agency for AIDS and Hepatitis Research VAC16 trial). The vaccine was able to induce HIV-specific CD4+ and CD8+ responses independently of the administration route, intramuscular or intradermal [100]. However, the intradermal route induced a weaker CD4+ response [100]. In summary, lipogenic biocarriers represent an interesting approach for future vaccines, not only in HIV, but with several other pathogens. Mucosal immunization Inducing effective long-lasting immunity in the mucosa would prevent virus entry and systemic infection. Considering that only some HIV quasi-species are infective through the mucosa, and that 80% of heterosexuals are infected by a single founder/transmitter virus [23, 101], a mucosal antibody response could be effective to this reduced infecting virus repertoire [102]. The site of vaccine application need not be the site of immunity induction, and it might alter cellular and humoral responses. In rhesus macaques, peroral administration of a SIV vaccine regimen was as effective as vaginal administration in producing vaginal IgAs and more effective than nasal, intestinal or vaginal routes in producing rectal IgAs [103]. In this study, immunization in the oral cavity elicited the most significant and diverse T cell responses as compared to multiple mucosal sites [103]. Nasal immunization may elicit widely disseminated mucosal IgA responses and greater IgG systemic responses than the peroral, rectal or vaginal routes. In an additional study, systemic immunization with SIV Gag, either through a DNA/NYVAC or recombinant BCG/NYVAC prime-boost strategy elicited potent and durable GI mucosal CD8+ responses. These cells were recruited from the systemic circulating pool early after vaccination and were mostly of the effector memory phenotype [104]. Thus, strong cellular immunity may be achieved at the mucosa through systemic vaccination. Cellular mucosal immunity is impaired by IL-13. Recently, a cytokine trap mucosal vaccine has shown to enhance the mucosal CD8+ response by incorporating soluble and membrane bound IL-13 receptor a2 to blocking the cytokine [105]. Nonetheless, vaccines capable of inducing immunity at the mucosal sites may not trigger systemic immunity and vice versa. Recently, a virosome harbouring surface HIV-1 lipidated gp41 P1 peptide (MYM-V101) was shown in a Phase I trial to be able to elicit, after intramuscular priming and intranasal booster doses, vaginal IgAs in 63% and rectal IgAs in 29% of women; while these antibodies were not Ó 2014 John Wiley & Sons Ltd

8 8 HIV Vaccines D. Lema et al. found to be neutralizing, they were shown to be able to inhibit virus transcytosis [106]. This effort could be the beginning of a new class of vaccines for several pathogens. Conclusions HIV vaccine development has proven to be particularly difficult in view of the evasive nature of the virus to the natural and artificially induced immune response attempting infection control. The uncooperativeness of the virus and the reductionist thinking have prevented researchers to reach greater advancements in HIV vaccine development [3]. A common drawback is the complexity of the immune response elicited by a vaccine and the scope of protection. The scope is often set at the molecular level, not taking into account important in vivo elements, often forgotten, until the vaccine is tried in animals. Antigen and immunogen, two different concepts, are often misinterpreted. Early vaccine development was directed to obtain viral subunits to induce specific antibodies. However, efforts to produce specific antigens (reverse vaccinology) to induce antibodies against the viral subunits have failed due to many factors. The designed antigen may not elicit production of antibodies with the same neutralizing capacity as compared to the antibody template as these antibodies were not found to decrease viral load or enhance immune cell responses. Other molecular combinations, involving several epitopes and paratopes, structural or spacial structures involved in antibody recognition and cell activation should be considered. In summary, the empirical approach of vaccine development has proven to be very slow. There is a rising need of randomized controlled trials, ultimate test to assess whether a vaccine can prevent or ameliorate infection, seem far away. One possible approach is the use of adaptive trials [4]. This trial design allows modification in the ongoing study due to the continuous analysis of real-time acquired data. The strategy shortens the time of analysis, that is, prematurely stopping a study due to lack of efficacy or adapting the study to the results where efficacy is noted. Concomitant non-vaccine prevention modalities must be taken into account in new trials. The combined strategy could bridge important data, in vitro and in vivo, and hence accelerating vaccine development in a rational fashion. The important issue is to produce an effective vaccine, which will not only serve for this disease, but it will be the start of new developments for other diseases, which have been difficult to treat as HIV. Past trials have shown that vaccine-elicited humoral or cellular responses have been insufficient to control infection. Most probably a combination of both arms of the immune system will yield an effective vaccine. 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