Vaccination Of Macaques Research Articles

Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope.

Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen. Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

Live-attenuated measles virus vaccine targets dendritic cells and macrophages in muscle of nonhuman primates.

Although live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MV(EZ). Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMV(EZ)EGFP(1), rMV(EZ)EGFP(3), and rMV(EZ)EGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMV(EZ)EGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMV(EZ)EGFP(1) and rMV(EZ)EGFP(6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMV(EZ)EGFP(3) was functionally equivalent to the commercial MV(EZ)-containing vaccine. Intramuscular vaccination of macaques with rMV(EZ)EGFP(3) resulted in the identification of EGFP(+) cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV. Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicates in vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

HIV-1 conserved elements p24CE DNA vaccine induces humoral immune responses with broad epitope recognition in macaques.

To target immune responses towards invariable regions of the virus, we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 p24gag. This conserved element vaccine is designed to avoid decoy epitopes by focusing responses to critical viral elements. We previously reported that vaccination of macaques with p24CE DNA induced robust cellular immune responses to CE that were not elicited upon wild type p55gag DNA vaccination. p24CE DNA priming followed by p55gag DNA boost provided a novel strategy to increase the magnitude and breadth of the cellular immune responses to HIV-1 Gag, including the induction of strong, multifunctional T-cell responses targeting epitopes within CE. Here, we examined the humoral responses induced upon p24CE DNA or p55gag DNA vaccination in macaques and found that although both vaccines induced robust p24gag binding antibody responses, the responses induced by p24CE DNA showed a unique broad range of linear epitope recognition. In contrast, antibodies elicited by p55gag DNA vaccine failed to recognize p24CE protein and did not recognize linear epitopes spanning the CE. Interestingly, boosting of p24CE DNA primed animals with p55gag DNA resulted in augmentation of antibodies able to recognize p24gag as well as the p24CE proteins, thereby inducing broadest immunity. Our results indicate that an effectively directed vaccine strategy that includes priming with the conserved element vaccine followed by boost with the complete immunogen induces broad cellular and humoral immunity focused on the conserved regions of the virus. This novel and effective strategy to broaden responses could be applied against other antigens of highly diverse pathogens.

Long-term immunogenicity studies of formalin-inactivated enterovirus 71 whole-virion vaccine in macaques.

Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO4 adjuvant, or PBS as control. During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.

Correction: Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

Correction: Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus SIV239 vaccine enhances SIV-specific humoral and cellular immunity and improves protection against a heterologous SIVE660 mucosal challenge.

It remains a challenge to develop a successful human immunodeficiency virus (HIV) vaccine that is capable of preventing infection. Here, we utilized the benefits of CD40L, a costimulatory molecule that can stimulate both dendritic cells (DCs) and B cells, as an adjuvant for our simian immunodeficiency virus (SIV) DNA vaccine in rhesus macaques. We coexpressed the CD40L with our DNA/SIV vaccine such that the CD40L is anchored on the membrane of SIV virus-like particle (VLP). These CD40L containing SIV VLPs showed enhanced activation of DCs in vitro. We then tested the potential of DNA/SIV-CD40L vaccine to adjuvant the DNA prime of a DNA/modified vaccinia virus Ankara (MVA) vaccine in rhesus macaques. Our results demonstrated that the CD40L adjuvant enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV CD8 and CD4 T cell responses, significantly delayed the acquisition of heterologous mucosal SIV infection, and improved viral control. Notably, the CD40L adjuvant enhanced the control of viral replication in the gut at the site of challenge that was associated with lower mucosal CD8 immune activation, one of the strong predictors of disease progression. Collectively, our results highlight the benefits of CD40L adjuvant for enhancing antiviral humoral and cellular immunity, leading to enhanced protection against a pathogenic SIV. A single adjuvant that enhances both humoral and cellular immunity is rare and thus underlines the importance and practicality of CD40L as an adjuvant for vaccines against infectious diseases, including HIV-1. Despite many advances in the field of AIDS research, an effective AIDS vaccine that can prevent infection remains elusive. CD40L is a key stimulator of dendritic cells and B cells and can therefore enhance T cell and antibody responses, but its overly potent nature can lead to adverse effects unless used in small doses. In order to modulate local expression of CD40L at relatively lower levels, we expressed CD40L in a membrane-bound form, along with SIV antigens, in a nucleic acid (DNA) vector. We tested the immunogenicity and efficacy of the CD40L-adjuvanted vaccine in macaques using a heterologous mucosal SIV infection. The CD40L-adjuvanted vaccine enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV T cell responses and improved protection. These results demonstrate that VLP-membrane-bound CD40L serves as a novel adjuvant for an HIV vaccine.

Differential Blood and Mucosal Immune Responses against an HIV-1 Vaccine Administered via Inguinal or Deltoid Injection

Mucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24–180 versus 180–365 days). HIV-1-specific CD8+ T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17–24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24–180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time.Trial RegistrationClinicalTrials.gov NCT00076817

Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.

Polymorphisms and Interspecies Differences of the Activating and Inhibitory FcγRII of Macaca nemestrina Influence the Binding of Human IgG Subclasses

Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His(131)). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His(131). These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.

SHIV Antigen Immunization Alters Patterns of Immune Responses to SHIV/Malaria Coinfection and Protects against Life-Threatening SHIV-Related Malaria

Whether vaccination against a virus can protect against more virulent coinfection with the virus and additional pathogen(s) remains poorly characterized. Overlapping endemicity of human immunodeficiency virus (HIV) and malaria suggests that HIV/malaria coinfection frequently complicates acute and chronic HIV infection. Here we showed that vaccination of macaques with recombinant Listeria ΔactA prfA* expressing simian/human immunodeficiency virus (SHIV) gag and env elicited Gag- and Env-specific T-cell responses, and protected against life-threatening SHIV-related malaria after SHIV/Plasmodium fragile coinfection. SHIV antigen immunization reduced peak viremia, resisted SHIV/malaria-induced lymphoid destruction, and blunted coinfection-accelerated decline of CD4(+) T-cell counts after SHIV/malaria coinfection. SHIV antigen immunization also weakened coinfection-driven overreactive proinflammatory interferon-γ (IFNγ) responses and led to developing T helper cell 17/22 (Th17/Th22) responses after SHIV/malaria coinfection. The findings suggest that vaccination against AIDS virus can alter patterns of immune responses to the SHIV/malaria coinfection and protect against life-threatening SHIV-related malaria.

Macaque-tropic human immunodeficiency virus type 1: breaking out of the host restriction factors

Macaque monkeys serve as important animal models for understanding the pathogenesis of lentiviral infections. Since human immunodeficiency virus type 1 (HIV-1) hardly replicates in macaque cells, simian immunodeficiency virus (SIV) or chimeric viruses between HIV-1 and SIV (SHIV) have been used as challenge viruses in this research field. These viruses, however, are genetically distant from HIV-1. Therefore, in order to evaluate the efficacy of anti-HIV-1 drugs and vaccines in macaques, the development of a macaque-tropic HIV-1 (HIV-1mt) having the ability to replicate efficiently in macaques has long been desired. Recent studies have demonstrated that host restriction factors, such as APOBEC3 family and TRIM5, impose a strong barrier against HIV-1 replication in macaque cells. By evading these restriction factors, others and we have succeeded in developing an HIV-1mt that is able to replicate in macaques. In this review, we have attempted to shed light on the role of host factors that affect the susceptibility of macaques to HIV-1mt infection, especially by focusing on TRIM5-related factors.

DNA vaccines expressing conserved elements provide potent and broad immune responses

Background Immunodominance and sequence diversity are major hurdles in the development of effective HIV vaccines. We tested the hypothesis that a vaccine candidate composed of strictly Conserved Elements (CE) of the HIV proteome excluding the variable regions would help overcome problems of viral sequence diversity and potential negative effects of immunodominance. Seven CE were identified in p24. Vaccination of macaques with p55DNA failed to elicit cellular or humoral immune responses to the CE, while epitopes outside of the CE were immunogenic.

Prime-boost regimen potency and efficacy with alphavirus replicons (SIV antigen) in non-human primates challenged with low-dose intra-rectal SIVsmE660

Background Self-amplifying RNAs (replicons) of positive-strand viruses such as alphaviruses are potentially safe and useful vectors for delivering vaccine antigens. Recombinant alphavirus replicon particles (VRP), carrying the self-amplifying RNA, protects rhesus macaques against SHIVSF162P4 challenge when used in a prime-boost regimen. Methods Novartis VRPs are being further tested using a current state-of-the art physiologically relevant low-dose SIV virus swarm challenge. To meet the need for the large numbers of VRP an alphavirus packaging cell line (PCL) was used for VRP production. We manufactured, characterized, stability and small animal potency tested VRPs expressing SIVmac239 envelope (env) and gag/pol fusion proteins (VRP Env, VRP Gag/pol, respectively) for a large macaque vaccine study. Macaques were co-immunized with both VRPs thrice followed by two boosts with an MF59-adjuvanted CHO cell-derived SIVmac239 trimeric env protein. Results

Immunogenicity of repeated Sendai viral vector vaccination in macaques

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8+ T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8+ T-cell responses specific for simian immunodeficiency virus Gag206–216 and Gag241–249, which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag206–216-specific and Gag241–249-specific CD8+ T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8+ T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.

Attenuated NYCBH vaccinia virus deleted for the E3L gene confers partial protection against lethal monkeypox virus disease in cynomolgus macaques

The New York City Board of Health (NYCBH) vaccinia virus is the currently licensed vaccine for use in the US against smallpox. The vaccine under investigation in this study has been attenuated by deletion of the innate immune evasion gene, E3L, and shown to be protective in homologous virus mouse challenge and heterologous virus mouse and rabbit challenge models. In this study we compared NYCBH deleted for the E3L gene (NYCBHΔE3L) to NYCBH for the ability to induce phosphorylation of proinflammatory signaling proteins and the ability to protect cynomolgus macaques from heterologous challenge with monkeypox virus (MPXV). NYCBHΔE3L induced phosphorylation of PKR and eIF2α as well as p38, SAPK/JNK, and IRF3 which can lead to induction of proinflammatory gene transcription. Vaccination of macaques with two doses of NYCBHΔE3L resulted in negligible pock formation at the site of scarification in comparison to vaccination using a single dose of NYCBH, but still elicited neutralizing antibodies and protected 75% of the animals from mortality after challenge with MPXV. However, NYCBHΔE3L-vaccinated animals developed a high number of secondary skin lesions and blood viral load similar to that seen in unvaccinated controls. The NYCBHΔE3L-vaccinated animals that survived MPXV challenge were able to show resolution of blood viral load, a decrease in number of skin lesions, and an improved clinical score by three weeks post challenge. These results suggest that although the highly attenuated NYCBHΔE3L allows proinflammatory signal transduction to occur, it does not provide full protection against monkeypox challenge.

Genomic Analysis Reveals Pre- and Postchallenge Differences in a Rhesus Macaque AIDS Vaccine Trial: Insights into Mechanisms of Vaccine Efficacy

We have employed global transcriptional profiling of whole blood to identify biologically relevant changes in cellular gene expression in response to alternative AIDS vaccine strategies with subsequent viral challenge in a rhesus macaque vaccine model. Samples were taken at day 0 (prechallenge), day 14 (peak viremia), and week 12 (set point) from animals immunized with replicating adenovirus type 5 host range (Ad5hr) recombinant viruses expressing human immunodeficiency virus HIV(env)(89.6P), simian immunodeficiency virus SIV(gag)(239), or SIV(nef)(239) alone or in combination with two intramuscular boosts with HIV(89.6P)gp140ΔCFI protein (L. J. Patterson et al., Virology 374:322-337, 2008), and each treatment resulted in significant control of viremia following simian-human immunodeficiency virus SHIV(89.6P) challenge (six animals per group plus six controls). At day 0, 8 weeks after the last treatment, the microarray profiles revealed significant prechallenge differences between treatment groups; data from the best-protected animals led to identification of a network of genes related to B cell development and lymphocyte survival. At peak viremia, expression profiles of the immunized groups were extremely similar, and comparisons to control animals reflected immunological differences other than effector T cell functions. Suggested protective mechanisms for vaccinated animals included upregulation of interleukin-27, a cytokine known to inhibit lentivirus replication, and increased expression of complement components, which may synergize with vaccine-induced antibodies. Divergent expression profiles at set point for the immunized groups implied distinct immunological responses despite phenotypic similarities in viral load and CD4(+) T cell levels. Data for the gp140-boosted group provided evidence for antibody-dependent, cell-mediated viral control, whereas animals immunized with only the replicating Ad5hr recombinants exhibited a different evolution of the B cell compartment even at 3 months postchallenge. This study demonstrates the sensitivity and discrimination of gene expression profiling of whole blood as an analytical tool in AIDS vaccine trials, providing unique insights into in vivo mechanisms and potential correlates of protection.

Role of nonhuman primates in the evaluation of candidate AIDS vaccines: an industry perspective

To consider how nonhuman primate (NHP) model systems can best contribute to HIV vaccine development. We review the traditional roles of NHP model systems in vaccine development and compare this with how NHP models have been used in HIV vaccine research and development. Comparisons of the immune responses elicited by cellular immune response-inducing vaccines in macaques and humans illustrate the value of primate studies for the relative ranking of HIV vaccine concepts for their likely immunogenicity in humans. The unusual structures (e.g. long complementarity-determining regions) of known broadly neutralizing HIV antibodies (bNAbs) suggest that it is critical to test candidate env immunogens in NHPs, whose germline antibody repertoires resemble those of humans. Recent clinical efficacy trial results question the utility of existing NHP challenge models in predicting HIV vaccine efficacy in humans, and highlight the need to further develop models in which acquisition of infection can be reliably evaluated. When evaluated in models using low virus dose challenges that better approximate human sexual exposure to HIV - some vaccine and passive NAb interventions appear to protect against acquisition of infection. NHP models have important roles in the preclinical evaluation, optimization, and ranking of novel HIV immunogens. The apparent vaccine efficacy observed using low virus dose challenge models provides an opportunity to investigate the correlates of protection.

Comparative Analysis of Immune Responses Induced by Vaccination With SIV Antigens by Recombinant Ad5 Vector or Plasmid DNA in Rhesus Macaques

DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.

25 years after discovering HIV as the cause of aids: prospects for a vaccine

Live attenuated HIV vaccines cannot be tested in humans because of their danger. Inactivated virions are still dangerous and also poorly immunogenic. For that reason subunit vaccines in one form or another have been the approach of almost all investigators since 1984 when adequate HIV culture systems became available. With the exception of using small animals for testing immune responses all in vivo experimental vaccine testing research utilize subhuman primates, especially macaques, challenged with SIV or the chimeric SHIV (using an HIV envelope with SIV). Thus, we begin with two handicaps -- a limitation to subunit vaccines and a limitation in our animal model. However, the limits of the animal model in my view are not due to any inadequacy of the model but rather the limitation of monkeys. This has been a problem from the beginning, but is one that could have been overcome long ago with adequate investments from funders. Instead, we witness most quality investigators limited in their experiments to 4 to 6 animals per group, so as to require multiple repeated experiments and years of delay, while a few groups can afford 20 to 30 animals per group. Obviously, this needs to change. A third handicap is HIV variation. Generally, investigators have challenged macaques with homologous SIV (or SHIV), but this is inadequate. However, in recent years the need for heterologous viral challenges has finally been recognized. It would be ideal to challenge with several different heterologous challenges, but there are serious limitations in available primate viral stocks, particularly SHIV. The best protocol for testing a candidate vaccine in macaques is controversial. Many have favored a single relatively high dose challenge, while recently several groups argue for multiple low dose challenges. The fact is arguments can be made for either, and no one knows which one will be more predictable for a HIV vaccine in man. By far the greatest hurdle to an effective vaccine is the retrovirus characteristics of HIV, namely its capacity to rapidly integrate its genes into our DNA. This establishes life-long infection, rapid diminished function of immune responses, and the emergence of variants. This feature has major implications all too often forgotten in the 1984 to 2008 period of HIV vaccine research, namely an effective HIV vaccine: (1) must protect against infection not just reduce HIV after infection; (2) must be long lasting; and (3) must be broad. Early in HIV vaccine history (roughly 1984-1990) the candidate immunogen was focused on the gp120 envelope delivered as protein, DNA, or vector with an HIV env gene insert. Mostly these led to type specific immune responses so vaccines of this type failed in the monkey model when challenges were made with heterologous strains of SIV or SHIV, especially with protein alone. Nonetheless, a company (VaxGen) still went forward with a gp120 protein clinical trial, and all too predictably, completely failed, and caused much negativity for HIV vaccine research. Many investigators then turned to CMI based vaccines. Though CMI is important and likely helpful to any vaccine candidate, CMI alone is a predictable failure, and indeed such vaccines have failed (e.g., recent NIAID -- Merck trials in Africa). We and some other groups think the answers will be in finding, fixing, and properly presenting conserved sequences of gp120 which are functionally required for HIV infection. Finding conserved sequences is an obvious need and a readily achievable one. Fixing means making a constrained envelope because the mobile envelope is a difficult and ever changing target. We have chosen the CCR5 binding domains of gp120 as the conserved and functionally necessary region. This region of gp120 contains novel epitopes called CD4i epitodes and Abs to them are known as CD4i Abs. We approach this by linking gp120 to domains of CD4 that bind gp120. We have developed a candidate vaccine based on inducing CD4i Abs. These Abs are often broadly reactive, and although we have not as yet reached sterilizing immunity, the vaccine ultimately protects against a high dose heterologous challenge and often has ADCC activity [1]. From our primate experiments and our studies in human elite viral suppressors [2] we predict that a successful HIV vaccine may consist of an immunogen that induces CD4i Abs and ADCC type responses.

P18-08. Characterization of CD34+ derived dendritic cells generated in vitro and transfected with HIV gene as potential therapeutic vaccine in macaque

Background Antiretroviral therapies against HIV infection reduce the plasma viral load but can not eradicate the virus. Dendritic cells (DC) transfected with messenger ribonucleic acids (mRNA) encoding endogenous viral proteins are expected to enhance the HIV specific immune response and are considered as potent therapeutic vaccines. In this context we studied the feasibility and efficiency of mRNA loaded CD34+ derived DC as therapeutic vaccine in SIV infected macaques as a model of HIV infection and AIDS.