– Thomas Harrer –
Although great progress has been achieved in the field of treatment and prevention of HIV-1 infection, the HIV-1 pandemic will ultimately be controlled only by an effective HIV-1 vaccine. Unfortunately, despite intense research for over two decades, it has not yet been possible to develop an effective protective HIV-1 vaccine. The following chapter will give a short overview of the current status of HIV-1 vaccine development.
Induction of neutralizing antibodies
Similar to successful vaccination strategies in other infections such as hepatitis B, initial HIV vaccine research focussed on the development of vaccines with the capability of inducing neutralizing antibodies. A variety of studies examined the safety and efficacy of vaccines such as gp120, gp160, parts of gp160 and peptides of gp160 to induce antibodies against HIV-1 envelope proteins. These immunogens stimulated the production of specific antibodies that were able to neutralize HIV-1 strains in vitro, but they failed to induce broadly neutralizing antibodies in HIV-1 variants derived from patients (Mascola 1996).
Two gp120-based vaccines were tested in two large Phase III trials in healthy volunteers: a clade B gp120 from HIV-1 MN and a gp120 from the CRF01_AE HIV-1 isolate were used in the VAX 003 Study in Thailand (Pitisuttithum 2006), while clade B gp120 proteins from HIV-1 MN and HIV-1 GNE8 were tested in the VAX 004 Study in the USA and the Netherlands (Flynn 2005). Despite induction of antibodies against gp120, the incidence of new infections was not lowered in either trial. These studies and others demonstrate that it is difficult to neutralize the biological activity of the envelope molecule gp160 via antibodies. Prior to the binding of gp120 to the CD4 receptor, the conserved and functionally important epitopes are hidden in grooves of the gp120 molecules that are additionally masked by glycan shields and variable sequence loops (Kwong 2002). Therefore, it is difficult for antibodies to block the binding of gp120 to the CD4 molecule.
The binding of the gp120 trimer to CD4 induces a conformational change of the V3 loop that exposes a conserved high-affinity coreceptor binding site on the gp120 molecule. The subsequent binding to the coreceptors CCR5 or CXCR4 triggers structural modifications of the viral transmembrane molecule gp41 and starts the fusion of the virus with the host cell membrane. Antibodies against the V3 loop can neutralize the process, although these activated binding sites on the V3 loop are recognizable by antibodies only for a short period of time. Therefore, high antibody concentrations are required for an efficient neutralization. Another problem for antibody-mediated neutralization is the shielding of the V3 loop-coreceptor interaction site by the gp120 trimer, which also inhibits the binding of antibodies to the V3 loop (Labrijn 2003).
HIV-1 infected patients generate neutralizing antibodies. However, in the majority of patients they are directed against the gp120 variable sequences. Due to the high sequence variability in gp120, HIV-1 can evade antibodies by a rapid generation of escape mutants. Thus, the majority of patients generate antibodies recognizing their own strain of HIV-1, but they neutralize HIV-1 variants from other patients poorly. There are only a few patients able to produce broadly cross-reacting neutralizing antibodies. These exceptional antibodies recognize the conserved binding sites for CD4 and coreceptors in gp120 and an important fusion domain in gp41. Vaccination with a recombinant gp120 molecule is not able to induce such antibodies as the V3 loop epitopes in the native gp120 molecule are not accessible to antibodies. To improve the induction of antibodies targeting the V3 loop, attempts are currently in progress to develop fusion molecules consisting of gp120 and CD4 that simulate the conformational changes in gp120 after binding to the CD4 molecule (Kwong 1998).
An innovative approach is the passive genetic immunization by the transfer of genes encoding highly active neutralizing antibodies or antibody-like immunoadhesins. In Rhesus monkeys, the intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding such SIV-specific antibody genes could induce the in vivo production of SIV-envelope-specific neutralizing antibody constructs that provided protection from intravenous challenge with SIV (Johnson 2009). This new exciting development stimulated a worldwide search for those few HIV-1 infected individuals that were able to generate unique highly active neutralizing antibodies that could be used for genetic immunization against HIV-1.
Induction of HIV-1-specific T cells
With all these hurdles regarding the induction of neutralizing antibody responses, the focus of vaccine development turned to vaccines that could elicit HIV-1-specific T cell responses. Cytotoxic T cells (CTL) play an important role in the control of HIV-1 in humans (Koup 1994, Harrer 1996b, Pantaleo 1997) and also for the control of SIV in SIV models. Experimental depletion of CD8 T cells in SIV-infected monkeys abrogated immune control of SIV infection and was associated with a strong increase of viral replication (Schmitz 1999). In contrast to neutralizing antibodies, CTLs do not exert a sterilizing immunity as they can only recognize cells that are already infected.
However, the observation of HIV-1-specific CTLs in HIV-1 exposed but uninfected subjects raised the hope that a T cell-based HIV-1 vaccine could prevent an ongoing HIV-1 infection by containment and eradication of small foci of viral infection (Herr 1998, Rowland-Jones 1998). Even if a T cell-based vaccine could not prevent infection of the host, there is the chance that it could influence the course of infection by reducing the extent of viremia after infection, as seen in the SIV monkey models (Letvin 2006). The viral load four months of infection, also known as the viral setpoint, may be one of the most important prognostic parameters for the course of HIV-1 infection. A vaccine could provide a clinical benefit if it could reduce the viral setpoint by half a log (Johnston 2007). In addition, such a vaccine could possibly exert positive effects on the spread of the HIV epidemic, as a lower viremia probably diminishes the infectivity of the patients. The clinical evaluation of these vaccines that do not prevent infection, but rather influence the course of disease, is difficult to achieve as large numbers of patients must be followed for extended periods of time.
HIV-1 can evade CTL recognition via development of CTL escape mutants in T cell epitopes or in proteasome cleavage sites (Maurer 2008). At least in conserved proteins such as gag or protease CTL-mediated immune selection is a major driving force for the development of polymorphisms (Mueller 2007). Our observations in long-term non-progressors showed that the quality of the CTL response with recognition of conserved CTL epitopes is very important (Harrer 1996a, Wagner 1999). It is essential for an effective vaccine to contain enough highly conserved CTL epitopes for the individual HLA alleles.
CTLs can be induced only by vaccines that are able to load viral peptides on HLA class I molecules of dendritic cells that present these peptides to the CTLs. Live attenuated viruses are effective against several infectious pathogens such as measles and they were protective against SIV in rhesus monkeys, but they are unlikely to be used in humans due to safety concerns. DNA vaccines alone are not very immunogenic in humans, but in DNA prime/vector boost strategies DNA priming could increase the immunogenicity of subsequent vaccinations with viral vectors. Lipopeptides allow the induction of CTL, but they can present only a limited repertoire of epitopes.
A new concept is the genetic immunization by transfer of genes encoding highly effective HIV-1-specific T-cell receptors (TCR) into CD8+ cytotoxic T-cells. In contrast to the transfer of antibody genes, transfer of TCR has to consider the HLA-restriction of the targeted CTL epitope and the HLA-I-type of the recipient. Recently, it could be shown in in-vitro experiments, that it is even possible to transfer two different exogenous HIV-1-specific TCRs into the same cell. If such techniques could be applied also in vivo, this could reduce the risk of selection of CTL escape mutations (Hofmann 2011).
Recombinant viral vectors
Recombinant viral vectors can achieve the induction of CTLs without the safety risks of attenuated live viruses. Several vectors have been tested in clinical studies: Adenovirus 5 (Ad5) vectors, ALVAC canarypox viruses, MVA (modified Vaccinia Virus Ankara), NYVAC (Gomez 2007a+b), adenovirus-associated virus and fowlpox vectors.
A great disappointment was the termination of two placebo-controlled Phase IIb trials, the HVTN 502 study (STEP trial) (Buchbinder 2008) and the HVTN 503 study (Phambili Study) (www.stepstudies.com). Both studies were testing Merck’s trivalent MRK Ad5 vaccine (V520), a mixture of Ad5 vectors expressing HIV-1 gag, pol and nef. 3000 volunteers from North America, South America, the Caribbean, and Australia participated in the STEP trial that started in December 2004. The vaccine was immunogenic and induced HIV-1-specific CD8+ T cells in 73% and HIV-1-specific CD4+ T cells in 41% of the vaccinees (McElrath 2008). Nevertheless, the study was terminated ahead of schedule in September 2007 because of lack of efficacy. The vaccine neither prevented HIV-1 infection nor did it lower the viral setpoint in those who were infected. 83 volunteers became infected during the trial. As only one female was infected, the post hoc analyses were restricted to the 82 male newly-infected subjects. There was a non-significant trend towards a greater number of infections in the vaccine recipients (49 new infections in 914 subjects) versus the placebo recipients (33 new infections in 922 subjects). Interestingly, subjects with high pre-existing Ad5-specific neutralizing antibody titers (titer of >200) at enrolment showed a higher infection rate in those who got the vaccine (21 infections) versus those in the placebo arm (9 infections). In contrast there were no significant differences in subjects with absent or low Ad5-specific neutralizing antibody titers of <200 (28 infections in the vaccine arm, 24 infections in the placebo arm). Because of the potential risk of the MRK Ad5 vaccine in subjects with a strong immune response against adenovirus 5, the parallel Phambili trial in South Africa was terminated as well. In Phambili, the MRK Ad5 vaccine showed no efficacy, with 33 new HIV-1 infections (4.54 infections per 100 person-years) in patients receiving at least one vaccination versus 28 HIV-1 infections (3.70 infections per 100 person-years) in the placebo arm (non-significant difference) (Gray 2011).
The STEP trial raises important questions that can be answered only by further examination of infected subjects and transmitted viruses. The fact that the increased infection risk was only seen in subjects with high antibody titers against the Ad5 vector argues against a general risk of immunizing against HIV-1, but it demonstrates the important issue of pre-existing vector immunity. The optimal priming of the immune response by a vaccine seems to be a key element determining the success or failure of a vaccine. Therefore more basic research is needed for a better understanding of the mechanisms of HIV-1 immunological control. Because of the unfavourable effects of pre-existing immunity against the adenovirus 5-vector, other adenoviral vectors are currently developed from less frequent adenovirus serotypes. So far, two phase-1 studies in healthy volunteers could demonstrate the immunogenicity of new HIV-1 vaccines based on the adenovirus serotypes AD26 (AD26.ENVA.01) and AD35 (AD35-GRIN/ENV) (Keefer MC 2010; Barouch D 2010).
In contrast to the STEP trial, the recently published RV144 study (Rerks-Ngarm 2009) involving more than 16,000 volunteers in Thailand showed a modest protective effect with a significant reduction of new HIV-1 infections by about 31%. The vaccine was a Sanofi Pasteur’s canarypox vector-based ALVAC HIV (vCP1521) expressing HIV-1 subtype B gag and protease and subtype E envelope in combination with AIDSVAX B/E gp120 proteins (MN rgp120/HIV-1 plus A244 rgp120/HIV-1). Among the 8198 subjects receiving placebo, 74 new HIV-1 infections were observed during the three years follow-up in comparison to 51 infections among the other half of volunteers that had received four immunizations with the ALVAC HIV and two immunizations with AIDSVAX B/E gp120 glycoproteins within a six months period. The vaccine had no effect on viral setpoints in the subjects infected despite vaccination, probably due to the fact that the vaccine induced only gp120-specific CD4 T cells (in 33% of the vaccinees), but almost no gag-specific CD4 T cells (in 1% of vaccines) and no HIV-1-specific CD8 T cells (measured by intracellular cytokine staining ICS). In contrast, almost every vaccinee developed high titer antibodies, although these antibodies only had a weak to moderate capacity to neutralize various HIV-1 strains. The mechanisms of the protective effect of the vaccine are still unresolved and it has been hypothesized that antibody-dependent cellular cytotoxicity (ADCC) may have played a role.
Another efficacy trial, the HVTN 505 study, started enrolment in 2009. This study is testing a prime boost vaccination regimen using DNA and a rAd5 vector containing env/gag/pol/nef.
A promising approach for the development of more effective HIV-1 vaccines is the therapeutic immunization of HIV-1-infected patients on ART who then undergo a treatment interruption (Harrer 2005). The analysis of a vaccine’s ability to control HIV-1 replication during treatment interruption may be a good instrument in identifying vaccines that are also effective in prevention.
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