猴免疫缺陷病毒外膜糖蛋白基因序列重组和突变克隆的转染研究

目的　研究猴免疫缺陷病毒 (SIV)外膜糖蛋白 (envGP)基因片段与病毒复制功能的关系。方法　利用两病毒株共有的内切酶位点 ,将非致病性SIVmac 142株envGP区的DNA片段与致病性SIVmac 2 39株的对应区域进行置换 ,构成多个重组体。RFLP和部分序列测序确定后 ,用等量突变病毒 (3μg)转染CD4 T淋巴细胞CEM× 174细胞系 ,用ELISA监测培养液中SIV核心蛋白P2 7水平的变化 ,以判定重组病毒的复制能力。结果　与SIVmac 2 39株相比 ,SIVmac 2 39envGP重组体 (SIV mac142 /2 39envGP/142 )仍保持高度的复制活性 ,SIVmac2 39gp41(SIVmac142 /2 39gp41/142 )或SIV mac2 39N gp41(SIVmac142 /2 39N gp41/142 )的复制能力明显降低 ,而SIVmac2 39C gp41(SIVmac142 /2 39C gp41/142 )重组体无复制活性。结论　envGP基因是SIVmac2 39株复制能力或毒力的重要调节因素。     

DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells

Dendritic cells (DCs) are essential for the early events of human immunodeficiency virus (HIV) infection. Model systems of HIV sexual transmission have shown that DCs expressing the DC-specific C-type lectin DC-SIGN capture and internalize HIV at mucosal surfaces and efficiently transfer HIV to CD4+ T cells in lymph nodes, where viral replication occurs. Upon DC-T cell clustering, internalized HIV accumulates on the DC side at the contact zone (infectious synapse), between DCs and T cells, whereas HIV receptors and coreceptors are enriched on the T cell side. Viral concentration at the infectious synapse may explain, at least in part, why DC transmission of HIV to T cells is so efficient.Here, we have investigated the role of DC-SIGN on primary DCs in X4 HIV-1 capture and transmission using small interfering RNA-expressing lentiviral vectors to specifically knockdown DC-SIGN. We demonstrate that DC-SIGN- DCs internalize X4 HIV-1 as well as DC-SIGN+ DCs, although binding of virions is reduced. Strikingly, DC-SIGN knockdown in DCs selectively impairs infectious synapse formation between DCs and resting CD4+ T cells, but does not prevent the formation of DC-T cells conjugates.Our results demonstrate that DC-SIGN is required downstream from viral capture for the formation of the infectious synapse between DCs and T cells. These findings provide a novel explanation for the role of DC-SIGN in the transfer and enhancement of HIV infection from DCs to T cells, a crucial step for HIV transmission and pathogenesis.     

Balance of cellular and humoral immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV infection

A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.There are formidable difficulties for developing a vaccine against a retrovirus such as HIV because of the integration of its genes into the DNA of the host target cells upon infection. For HIV, this problem is compounded by HIV-induced immune suppression and the development of variants that escape immune control. Consequently, an effective preventive vaccine against HIV must work early to block HIV infection and quickly kill HIV-infected cells, or both. To date, only antibodies to the HIV envelope glycoprotein (Env) fit this requirement. Available evidence suggests that such antibodies must recognize highly conserved domains and could inhibit infection by direct neutralization or by Fc receptor-dependent effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC) (1, 2). The ideal result would be sterilizing immunity or, at a minimum, a major restriction of the infection (3). Another challenge stems from evolutionary pressures that abrogate the immunogenicity of conserved, functional epitopes on the envelope spike that are potential targets for cross-reactive antibodies. Large areas are masked by a “glycan shield” of carbohydrate molecules and extensive conformational flexibility (sometimes termed “conformational masking”) that dampen immunogenicity of the conserved functional domains (4, 5). The remaining immunogenic domains (“variable” or “V” loops) tolerate a high degree of sequence variability and generate “type-specific” neutralizing antibodies that are not cross-reactive and that limit the efficacy of vaccines that use conventional gp120 monomeric protein.An emerging concern for HIV vaccine development centers on the quantitative and qualitative aspects of T-cell activation elicited by various immunization regimens (6). Although HIV-specific T cells might potentially combat infection, certain patterns of T-cell activation (e.g., involving CD4+ CCR5+ T cells) have the potential to promote HIV replication. The latter possibility is emphasized by the HIV vaccine-associated increased risk of infection seen in two large human clinical trials that selectively generated HIV-specific T-cell responses (7). Similar associations between increased risk of infection and T-cell responses of various sorts have been reported in the nonhuman primate model (8–10). Thus, the ideal HIV vaccine strategy is likely to be one that generates antiviral humoral responses without incurring T-cell activation profiles that promote infection and/or overcome the protective benefits of antibodies. Insights for such an approach can be gained by comparative analyses of nonhuman primate models of HIV infection.The vaccine concept that we have been testing is designed to overcome some of these challenges by stably expressing a highly conserved transition state structure that is exposed on gp120 during a key step in viral entry, exposure of the coreceptor-binding domain consequent to CD4 binding. The prototype immunogen [full-length single chain (FLSC)] is a chimeric protein composed of gp120 from the HIV-1_Ba-L isolate fused to the N terminus of the two outer domains of CD4 by a flexible polypeptide linker (11). For studies of rhesus macaques, the construct is modified to contain “self” rhesus macaque CD4 sequences (rhFLSC) to avoid anti-CD4 responses. The rhFLSC elicits antibody responses to highly conserved epitopes, including the coreceptor-binding domain epitopes (CoRBS) and the C1 regions implicated as a potent ADCC target (12). In an earlier study (12), we showed that rhesus macaques vaccinated with rhFLSC formulated with QS21, a saponin adjuvant derived from the soap-bark tree Q. saponaria, exhibited accelerated clearance of plasma viremia and an absence of long-term tissue viral loads compared with unvaccinated controls after a single high-dose rectal challenge with heterologous SHIV162P3. Postinfection control correlated with stronger responses to CD4i epitopes in the rhFLSC-vaccinated animals (CD4i titers > 1:100), compared with macaques that received control immunogens including gp120, soluble CD4, or chemically cross-linked gp120-CD4. Postinfection control did not correlate with anti-CD4 responses, overall anti-gp120–binding titers, or neutralizing activity measured in conventional assays (12), although it did correlate with neutralizing titers in the soluble CD4-triggered assay using HIV-2_7312A/V434M that selectively detects responses to highly conserved epitopes in the coreceptor-binding site (13). Taken together, this study showed that rhFLSC elicits antibody responses to highly conserved CD4i epitopes that correlate with postinfection control of viremia after a high-dose rectal challenge with SHIV162P3, but it left open the question of whether rhFLSC can elicit antibodies that block acquisition. Acquisition is typically blocked only in high-dose challenge studies when the vaccine and challenge stock are matched (14), which is not the case for rhFLSC and SHIV162P3. For this reason, we performed three independent studies using different rhFLSC immunization schemes and a repeat low-dose rectal challenge model that is thought to be more reflective of sexual HIV transmission (15). These studies were designed in part as a hypothesis-generating exercise with respect to protective immunity. We consistently found (i) inverse correlations between acquisition of infection and certain aspects of humoral immunity and (ii) direct relationships between acquisition of infection and vaccine-elicited T-cell responses. Importantly, in certain test groups the apparent protective benefit of humoral responses is absent when T-cell responses are comparatively high. These results strongly suggest that a successful HIV vaccine will need to elicit protective antibody responses without eliciting attenuating levels of vaccine-elicited T-cell responses.     

TRANSVAC research infrastructure – Results and lessons learned from the European network of vaccine research and development

TRANSVAC was a collaborative infrastructure project aimed at enhancing European translational vaccine research and training. The objective of this four year project (2009–2013), funded under the European Commission's (EC) seventh framework programme (FP7), was to support European collaboration in the vaccine field, principally through the provision of transnational access (TNA) to critical vaccine research and development (R&D) infrastructures, as well as by improving and harmonising the services provided by these infrastructures through joint research activities (JRA). The project successfully provided all available services to advance 29 projects and, through engaging all vaccine stakeholders, successfully laid down the blueprint for the implementation of a permanent research infrastructure for early vaccine R&D in Europe.     

Immunogenicity of newly constructed attenuated vaccinia strain LC16m8Δ that expresses SIV Gag protein

We developed the method to efficiently construct recombinant vaccinia viruses based on LC16m8Δ strain that can replicate in mammalian cells but is still safe in human. Immunization in a prime-boost strategy using DNA and LC16m8Δ expressing SIV Gag elicited 7–30-fold more IFN-γ-producing T cells in mice than that using DNA and non-replicating vaccinia DIs recombinant strain. As the previous study on the DNA-prime and recombinant DIs-boost anti-SIV vaccine showed protective efficacy in the macaque model [Someya K, Ami Y, Nakasone T, Izumi Y, Matsuo K, Horibata S, et al. Induction of positive cellular and humoral responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol. J Immunol 2006;176(3):1784–95], LC16m8Δ would have potential as a better recombinant viral vector for HIV vaccine.     

Longitudinal study to assess the safety and efficacy of a live-attenuated SHIV vaccine in long term immunized rhesus macaques

Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of ΔvpuSHIV_PPC, a live virus vaccine derived from SHIV_PPC. Macaques were administered two inoculations of ΔvpuSHIV_PPC, three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.     

Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques

We investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization.Both immunization strategies induced strong SIV Gag-specific IFN-γ and T-cell proliferation responses and mediated a conservation of CD4⁺ memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply.     

Control of viremia and maintenance of intestinal CD4 memory T cells in SHIV162P3 infected macaques after pathogenic SIVMAC251 challenge

Recent HIV vaccine failures have prompted calls for more preclinical vaccine testing in non-human primates. However, similar to HIV infection of humans, developing a vaccine that protects macaques from infection following pathogenic SIV_MAC251 challenge has proven difficult, and current vaccine candidates at best, only reduce viral loads after infection. Here we demonstrate that prior infection with a chimeric simian-human immunodeficiency virus (SHIV) containing an HIV envelope gene confers protection against intravenous infection with the heterologous, highly pathogenic SIV_MAC251 in rhesus macaques. Although definitive immune correlates of protection were not identified, preservation and/or restoration of intestinal CD4⁺ memory T cells were associated with protection from challenge and control of viremia. These results suggest that protection against pathogenic lentiviral infection or disease progression is indeed possible, and may correlate with preservation of mucosal CD4⁺ T cells.     

Differential patterns of immune escape at Tat-specific cytotoxic T cell epitopes in pigtail macaques

Cytotoxic T lymphocyte responses to conserved proteins such as Gag within HIV- or SIV-infected hosts can facilitate partial control of viremia. However, the utility of targeting variable viral proteins by CTL responses is unclear. We studied CTL responses to regulatory and accessory proteins of SIV in pigtail macaques. The regulatory and accessory proteins were the most commonly targeted proteins by CTL responses from pigtail macaques. We identified 2 novel Tat-specific CTL responses that were both restricted by the Mane-A?10 allele. Viral escape at one of the Tat epitopes, KSA10, was slower in comparison to another Tat epitope KVA10. The kinetics of escape of the KSA10 Tat epitope were more similar to an immunodominant KP9 Gag epitope also restricted by Mane-A?10. Our results suggest that some regulatory or accessory CTL epitopes may be useful targets for vaccination against HIV.