Development of rationally designed HIV vaccines

成果1：评估哪一种猕猴模型能更好地预测或概括HIV候选疫苗在人类中的功效。SIVmac251模型预测并概括了RV144: 2005年，一项针对新生儿的合作研究预测了RV144的结果。最近，我们总结了RV144疫苗方案在幼年（2-3岁）雄性和雌性猕猴中的有效性。后一项研究提供了比仅仅评估疫苗效力更有价值的东西。我们对PBMCs和外泌体中的RNA表达进行了综合研究，并对血液、淋巴结和粘膜中的先天和适应性免疫反应进行了全面分析，发现了意想不到的病毒获得风险相关因素，如粘膜先天淋巴样细胞（ILCs）和12个基因的表达，其中10个是RAS通路的一部分。我与Strbo博士和迈阿密大学已故的Eckhart Podack博士合作开发的另一种保护性疫苗方案的类似观察结果证实了该模型的有效性。这种方法是一项联合专利的主题。我们还进行了一项完全由NIAID艾滋病司支持的验证性研究，在其他动物设施中，我们比较了ALVAC和NYVAC基于siv的疫苗方案的免疫原性和有效性。基于ALVAC-SIV的疫苗对中国猕猴也有保护作用，而基于nyvac的“更具免疫原性”的SIV重组疫苗则没有。值得注意的是，基于nyvac的疫苗没有像非保护性Ad26初始方案那样诱导IL-1B的早期爆发。SIVmac251模型能预测hvt -702的结果吗？我们在SIVmac251模型中测试了基于alvac的SIV疫苗的有效性，在gp120增强中使用明矾与MF59佐剂并行。令我们惊讶的是，SIV模型中基于MF59的方案并不有效，尽管其免疫原性更高。虽然这一观察结果在科学界引起了争论，但我们的发现并没有影响P5伙伴关系在南非HVTN-702研究中使用MF59作为佐剂的决定。相反，后勤和政治因素可能影响了按原计划进行的决定。考虑到目前SIVmac251动物模型预测人类结果的准确性的不确定性，我们的研究将为定义该模型的效用提供关键信息，因为我们在猕猴中没有观察到有效性。hvt -702中无效的初步分析将于2018年公布，并将解决这一问题。雌性中国恒河猴的SHIV-clade C Tier 1：在另一项主要由NIAID支持的研究中，用HVTN702中使用的相同的基于alvac - hiv的疫苗对恒河猴进行免疫，在gp120增强剂中结合MF59或明铝（诺华），并用SHIV clade C Tier 1病毒进行攻击。以MF59为基础的方案具有保护作用，而alumN则没有。保护作用与SHIV分支C Tier 1病毒的中和抗体有关。在目前的状态下，我认为这个SHIV模型不能预测hvt -702的结果，因为在南非只有1%的HIV进化支C病毒对中和敏感。然而，对HVTN-702是否无效的中期分析将进一步加深我们对动物模型可预测性的认识，并将指导选择和改进可靠的动物模型，以开发完全有效的艾滋病毒疫苗。2. CD14+单核细胞缺氧和炎性体的激活与SIVmac251获得风险降低相关。该研究表明，CD14+和CD16+单核细胞在SIVmac251获得风险中起作用，并有助于了解IL-1B和IL-10在DNA/ALVAC/明矾疫苗平台中的作用机制。我们比较了ALVAC-SIV、DNA-SIV和Ad26-SIV引物方式与两种ALVAC-SIV + gp120蛋白增强在年轻雄性猕猴中的联合作用。我们使用肌内DNA- siv引物的目的是增加CD4+ T细胞和抗体反应，因为我们在之前的工作中证明了DNA引物诱导的CD4+ T细胞会影响抗包膜滴度。选择Ad26-SIV引物的目的是增加粘膜抗体对Env和CD8+ T细胞的反应。DNA引物在DNA引物方案中保护抗感染，而VE与经典CD14+ cd16阴性单核细胞的缺氧和炎性体激活相关，这反过来又与肠道归巢ccr5阴性Th2细胞反应直接相关，并与粘膜NKp44+细胞和IgG对V2直接相关。在同一组中，分化的CD16+单核细胞的频率通过STAT3激活与疫苗效力降低相关。Ad26主要方案不能防止SIV获得，尽管其免疫原性较高，并且与血液中CD14+ DR+单核细胞总数减少，淋巴结中新生分化的CX3CR1+ CD163+巨噬细胞积累，粘膜炎症以及直肠粘膜和肺部CD4+ T细胞向Th17细胞倾斜有关。Th17细胞优先被HIV/SIV感染，并负向调节NKp44+细胞。正如在其他针对猕猴和人类的疫苗研究中所观察到的那样，疫苗产生的用于病毒感染的靶细胞可能降低了Ad26初级方案的效力。3. 基于DNA/ALVAC-SIV /ALVAC-SIV + gp120 /明矾的简化疫苗方案可提供69%的疫苗效力。为了证实单核细胞介导的先天免疫的重要性，并研究DNA/ALVAC/gp120/明矾方法是否在雌性猕猴中也有效，我们设计了一项针对年轻和老年雌性猕猴的研究。我们测试了一种简化版本的DNA引物ALVAC/gp120/明矾疫苗方法，使用两次肌肉注射DNA引物（第0周和第4周），一次ALVAC- siv增强（第8周），以及在第13周使用ALVAC- siv和gp120在明矾中联合进行一次最终增强。挑战暴露于第17周开始，持续11周。我们发现该疫苗在年轻雌性猕猴（平均年龄2.7岁）中有69%的VE，但不能保护老年雌性猕猴（平均年龄7.5岁）。年轻女性的保护作用与CD14+经典单核细胞的频率相关，证实了先前在年轻男性中的研究结果。然而，在老年接种过疫苗的女性中，我们观察到血浆IL-6和IL-8水平与病毒获取增加相关。这些数据进一步支持单核细胞在雌性和雄性猕猴保护中的作用，并揭示了一个年龄依赖性因素。在年轻或年老的幼稚对照中，SIVmac251的获取没有差异（数据未显示）。研究现状及未来规划定义CD14+单核细胞、中性粒细胞和Th2 ccr5阴性T细胞的表观遗传特征，研究与保护性免疫相关的细胞外囊泡（EV）中的miRNA特征。2. 确定保护性疫苗诱导的v2靶向体液反应与先天免疫之间的特异性和机制交叉3。下一代基于ALVAC的HIV候选疫苗的开发利用先天免疫引发持久的保护性免疫特征研究抑制a4B7整合素结合的V2单克隆抗体是否单独或与先天单核细胞记忆免疫联合保护SIVmac251获得5。耐久性6。研究年龄偏倚疫苗效力的基础，并测试可能克服它的佐剂，如IGF-1和铝基脂质体ALFA佐剂。设计一种基于HIV进化支B的最佳DNA/ALVAC/gp120/明矾疫苗方案，并在年轻志愿者中进行I期HIV疫苗试验。我们还将试图了解单核细胞在COV-2感染中的作用。

Accomplishments 1: Assess which macaque model better predicts or recapitulates the efficacy of HIV vaccine candidates in humans. The SIVmac251 model has predicted and recapitulated RV144: In 2005, a collaborative study in neonates predicted the results of RV144. More recently we recapitulated the efficacy of the RV144 vaccine regimen in young (2-3 years) male and female macaques. The latter study has provided more value than just the evaluation of vaccine efficacy. Our integrated study of RNA expression in PBMCs and exosomes together with comprehensive analyses of innate and adaptive immune responses in blood, lymph nodes, and mucosa uncovered unexpected correlates of risk of virus acquisition such as mucosal innate lymphoid cells (ILCs) and the expression of 12 genes, ten of which are part of the RAS pathway. The usefulness of this model was validated by similar observations in another protective vaccine regimen that I developed in collaboration with Dr. Strbo and the late Dr. Eckhart Podack of Miami University. This approach was the subject of a joint patent. We have also performed a confirmatory study entirely supported by the Division of AIDS, NIAID, in other animal facilities wherein we compared the immunogenicity and efficacy of the ALVAC and NYVAC SIV-based vaccine regimens. The ALVAC-SIV based vaccine was also protective in Chinese macaques, whereas the "more immunogenic" NYVAC-based SIV recombinant vaccine was not. Of note, the NYVAC-based vaccine did not induce an early burst of IL-1B as was the case in the non-protective Ad26 prime regimen. Will the SIVmac251 model predict the results of HVTN-702? We tested the efficacy of the ALVAC-based SIV vaccines in the SIVmac251 model using the alum in parallel with the MF59 adjuvant in the gp120 boost. To our surprise, the MF59 based regimen in the SIV model was not efficacious, despite its higher immunogenicity. While this observation stirred debate in the scientific community, our findings did not influence the decision by the P5 Partnership to use MF59 as an adjuvant in the HVTN-702 study in South Africa. Instead, logistical and political factors likely influenced the decision to proceed as originally planned. Given the current uncertainty of how accurately the SIVmac251 animal model will predict human results, our study will provide crucial information to define the model's utility since we observed no efficacy in macaques. The preliminary analysis of futility in HVTN-702 will be made public in 2018 and will address this issue. SHIV-clade C Tier 1 in female Chinese rhesus macaques: In another study supported mostly by NIAID, macaques were immunized with the identical ALVAC-HIV-based vaccines used in HVTN702, combined with either the MF59 or the alum (Novartis) in the gp120 boost, and challenged with a SHIV clade C Tier 1 virus. The MF59 based regimen was protective, whereas the alumN was not. Protection was associated with neutralizing antibodies to the SHIV clade C Tier 1 virus. In its current state, I don't believe that this SHIV model will predict the results of HVTN-702 because only 1% of HIV clade C viruses in South Africa are sensitive to neutralization. However, the interim analysis of HVTN-702 for futility will further our knowledge of animal model predictability and will guide the choice and refinement of faithful animal models for the development of fully effective vaccines for HIV. 2. HIV vaccine candidate activation of hypoxia and inflammasome in CD14+ monocytes is associated with a decreased risk of SIVmac251 acquisition. This study demonstrated that CD14+ and CD16+ monocytes have a role in the risk of SIVmac251 acquisition and has contributed to the mechanistic insight on the role of IL-1B and IL-10 in the DNA/ALVAC/alum vaccine platform. We compared ALVAC-SIV, DNA-SIV, and Ad26-SIV prime modalities in combination with two ALVAC-SIV + gp120 protein boosts in young male macaques. We used the intramuscular DNA-SIV prime with the intent of increasing CD4+ T cells and antibody responses since we demonstrated in prior work that the CD4+ T cells elicited by the DNA prime influence the anti-envelope titers. The Ad26-SIV prime was chosen with the intent of increasing mucosal antibodies to Env and CD8+ T cell responses. The DNA prime protected against infection in the DNA prime regimen, and VE correlated with hypoxia and inflammasome activation in classical CD14+ CD16negative monocytes that in turn directly correlated with the gut-homing CCR5negative Th2 cell responses and with mucosal NKp44+ cells and IgG to V2. In the same group, the frequency of differentiated CD16+ monocytes correlated with reduced vaccine efficacy via STAT3 activation. The Ad26 prime regimen did not protect against SIV acquisition, despite its higher immunogenicity, and it was associated with a decrease in total CD14+ DR+ monocytes in blood, accumulation of de novo differentiated CX3CR1+ CD163+ macrophages in lymph nodes, mucosal inflammation, and skewing of CD4+ T cells toward Th17 cells in the rectal mucosa and lungs. Th17 cells are preferentially infected by HIV/SIV and negatively regulate NKp44+ cells. It is possible that vaccine-generated target cells for virus infection, may have decreased the efficacy of the Ad26 prime regimen, as was observed in other vaccine studies in macaques and humans. 3. A simplified vaccine regimen based on DNA/ALVAC-SIV / ALVAC-SIV + gp120 / alum affords 69% vaccine efficacy. With the intent of confirming the importance of monocyte-mediated innate immunity and investigating whether the DNA/ALVAC/gp120/alum approach was also effective in female macaques, we designed a study in young and old females. We tested a simplified version of the DNA prime ALVAC/gp120/alum vaccine approach using two intramuscular DNA primes (weeks 0 and 4), one ALVAC-SIV boost (week 8), and a single final boost with the combination ALVAC-SIV and gp120 in alum at week 13. Challenge exposure was started at week 17 and continued for 11 weeks. We found that this vaccine had 69% VE in young female macaques (average age 2.7 years), but failed to protect old female macaques (average age 7.5 years). Protection in young females correlated with the frequency of CD14+ classical monocytes, confirming the results of the previous study in young males. In old vaccinated females, however, we observed a correlation with the plasma level of IL-6 and IL-8 and increased virus acquisition. These data further support a role for monocytes in protection of both female and male macaques and uncovered an age-dependent factor. No difference in SIVmac251 acquisition was observed in young or old naive controls (data not shown). Current Research and Future Plans 1. Define the epigenetic signatures of CD14+ monocytes, neutrophils, and Th2 CCR5negative T cells and study the miRNA signatures in extracellular vesicles (EV) that track with protective immunity. 2. Define the specificity and mechanistic intersection between protective vaccine-induced V2-targeted humoral responses and innate immunity 3. Development of the next generation of ALVAC based HIV vaccine candidate leveraging innate immunity to elicit durable protective immune profiles 4. Investigate whether monoclonal antibodies to V2 that inhibit a4B7 integrin binding protect against SIVmac251 acquisition, alone or in combination with innate monocyte memory immunity 5. Durability 6. Investigate the basis for the age bias vaccine efficacy and test adjuvants that may overcome it, such as IGF-1 and an alum-based liposome ALFA adjuvant with respect to innate immunity. 7: Design an HIV clade B based optimal DNA/ALVAC/gp120/alum vaccine regimen and test it in a phase I HIV vaccine trial in young volunteers. We will also seek to understand the role of monocytes in COV-2 infection.