Cervico-Vaginal Inflammatory Cytokine and Chemokine Responses to Two Different SIV Immunogens.

Studies have shown that vaccine vectors and route of immunization can differentially activate different arms of the immune system. However, the effects of different HIV vaccine immunogens on mucosal inflammation have not yet been studied. Because mucosal sites are the primary route of HIV infection, we evaluated the cervico-vaginal inflammatory cytokine and chemokine levels of Mauritian cynomolgus macaques following immunization and boost using two different SIV vaccine immunogens. The PCS vaccine delivers 12 20-amino acid peptides overlapping the 12 protease cleavage sites, and the Gag/Env vaccine delivers the full Gag and full Env proteins of simian immunodeficiency virus. We showed that the PCS vaccine prime and boosts induced short-lived, lower level increases of a few pro-inflammatory/chemotactic cytokines. In the PCS-vaccine group only the levels of MCP-1 were significantly increased above the baseline (P = 0.0078, Week 6; P = 0.0078, Week 17; P = 0.0234; Week 51) following multiple boosts. In contrast, immunizations with the Gag/Env vaccine persistently increased the levels of multiple cytokines/chemokines. In the Gag/Env group, higher than baseline levels were consistently observed for IL-8 (P = 0.0078, Week 16; P = 0.0078, Week 17; P = 0.0156, Week 52), IL-1ß (P = 0.0234, Week 16; P = 0.0156, Week 17; P = 0.0156, Week 52), and MIP-1α (P = 0.0313, Week 16; P = 0.0156, Week 17; P = 0.0313, Week 52). Over time, repeated boosts altered the relative levels of these cytokines between the Gag/Env and PCS vaccine group. 18 weeks after final boost with a higher dosage, IP-10 levels (P = 0.0313) in the Gag/Env group remained higher than baseline. Thus, the influence of vaccine immunogens on mucosal inflammation needs to be considered when developing and evaluating candidate HIV vaccines.

Vaccine targeting SIVmac251 protease cleavage sites protects macaques against vaginal infection.

After over 3 decades of research, an effective anti-HIV vaccine remains elusive. The recently halted HVTN702 clinical trial not only further stresses the challenge to develop an effective HIV vaccine but also emphasizes that unconventional and novel vaccine strategies are urgently needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provided greater than 80% protection to Mauritian cynomolgus macaques against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses correlated with vaccine efficacy. The PCS vaccine did not induce immune activation or inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune microenvironment generated by the PCS vaccine was predictive of vaccine efficacy. Our study demonstrates, for the first time to our knowledge, that a vaccine which targets only viral maturation, but lacks full-length Env and Gag immunogens, can prevent intravaginal infection in a stringent macaque/SIV challenge model. Targeting HIV maturation thus offers a potentially novel approach to developing an effective HIV vaccine.

Polysaccharide Nanoparticles Can Efficiently Modulate the Immune Response against an HIV Peptide Antigen.

The development of an effective HIV vaccine continues to be a major health challenge since, so far, only the RV144 trial has demonstrated a modest clinical efficacy. Recently, the targeting of the 12 highly conserved protease cleavage sites (PCS1-12) has been presented as a strategy seeking to hamper the maturation and infectivity of HIV. To pursue this line of research, and because peptide antigens have low immunogenicity, we have included these peptides in engineered nanoparticles, aiming at overcoming this limitation. More specifically, we investigated whether the covalent attachment of a PCS peptide (PCS5) to polysaccharide-based nanoparticles, and their coadministration with polyinosinic:polycytidylic acid (poly(I:C)), improved the generated immune response. To this end, PCS5 was first conjugated to two different polysaccharides (chitosan and hyaluronic acid) through either a stable or a cleavable bond and then associated with an oppositely charged polymer (dextran sulfate and chitosan) and poly(I:C) to form the nanoparticles. Nanoparticles associating PCS5 by ionic interactions were used in this study as the control formulation. In vivo, all nanosystems elicited high anti-PCS5 antibodies. Nanoparticles containing PCS5 conjugated and poly(I:C) seemed to induce the strongest activation of antigen-presenting cells. Interestingly, T cell activation presented different kinetics depending on the prototype. These findings show that both the nanoparticle composition and the conjugation of the HIV peptide antigen may play an important role in the generation of humoral and cellular responses.

Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques.

HIV mutates rapidly and infects CD4+ T cells, especially when they are activated. A vaccine targeting conserved, essential viral elements while limiting CD4+ T cell activation could be effective. Learning from natural immunity observed in a group of highly HIV-1 exposed seronegative Kenyan female sex workers, we are testing a novel candidate HIV vaccine targeting the 12 viral protease cleavage sites (PCSs) (the PCS vaccine), in comparison with a vaccine targeting full-length Gag and Env (the Gag/Env vaccine) in a Mauritian cynomolgus macaque/SIV model. In this study we evaluated these vaccines for induction of mucosal antibodies to SIV immunogens at the female genital tract. Bio-Plex and Western blot analyses of cervicovaginal lavage samples showed that both the PCS and Gag/Env vaccines can elicit mucosal IgG antibody responses to SIV immunogens. Significantly higher increase of anti-PCS antibodies was induced by the PCS vaccine than by the Gag/Env vaccine (p<0.0001). The effect of the mucosal antibody responses in protection from repeated low dose pathogenic SIVmac251 challenges is being evaluated.

Hypothetical endogenous SIV-like antigens in Mauritian cynomolgus macaques.

Simian immunodeficiency virus (SIV) infection of Mauritian cynomolgus macaques (MCMs) is an increasingly important nonhuman primate model for HIV vaccine research. We previously reported that in MCMs anti-SIV antibodies can be naturally developed without exogenous infection or vaccination, and that a vaccine targeting SIV protease cleavage sites (PCS) can cross-induce antibodies to non-PCS SIV antigens. We speculate that this is potentially caused by the existence of endogenous SIV-like antigens. External stimuli (such as environmental factors and vaccination) may induce expression of endogenous SIV-like antigens to elicit these antibodies. Database and mass spectrometry analyses were conducted to search for such antigens. We identified endogenous SIV-like DNA sequences in cynomolgus macaque genome and non-PCS peptide homologous to SIV Env protein in PBMCs of a PCS-vaccinated monkey. Our preliminary insights suggest that endogenous SIV-like antigens may be one of the possible reasons for the natural and cross-inducible SIV antibodies in MCMs.

Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques.

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research.

A novel HIV vaccine targeting the protease cleavage sites.

HIV preferentially infects activated CD4+ T cells and mutates rapidly. The classical vaccine approach aimed to generate broad immune responses to full HIV proteins largely failed to address the potential adverse impact of increased number of activated CD4+ T cells as viral targets. Learning from natural immunity observed in a group of HIV resistant Kenyan female sex workers, we are testing a novel vaccine approach. It focuses immune response to the highly conserved sequences surrounding the HIV protease cleavage sites (PCS) to disrupt viral maturation, while limiting excessive immune activation. Our pilot studies using nonhuman primate SIV infection models suggest that this approach is feasible and promising.

Mauritian cynomolgus macaques with M3M4 MHC genotype control SIVmac251 infection.

BACKGROUND: Understanding natural HIV control may lead to new preventative or therapeutic strategies. Several protective major histocompatibility complex (MHC) genotypes were found in humans and rhesus macaques. Here, we report a simian immunodeficiency virus (SIV) controller MHC genotype in Mauritian cynomolgus macaques (MCMs). METHODS: Twelve MHC-genotyped MCMs were infected with SIVmac251 and monitored for viral loads and CD4+ T-cell counts. RESULTS: Two macaques with M3M4 genotype exhibited the lowest peak viral loads (log plasma SIV RNA copies/mL), nearly 3 logs lower than those in most macaques with other MHC haplotype combinations, and set point viral loads below the level of detection limit by RT-qPCR (<2 log RNA copies/mL). They maintained healthy CD4+ T-cell counts of >500 cells/µL blood, while CD4 counts in the vast majority of other macaques were below this level. CONCLUSIONS: The M3M4 MHC genotype may confer enhanced control of SIV replication in MCMs.