CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.     

Analysis of humoral immune responses in rhesus macaques vaccinated with attenuated SIVmac239Δnef and challenged with pathogenic SIVmac251

Background To determine the correlation between protection and humoral immune response against simian immunodeficiency virus (SIVmac251), 11 macaques were immunized with live‐attenuated SIVmac239Δnef either intravenously or via the tonsils and exposed to SIVmac251 after either 6 or 15 months along with unvaccinated controls. Results Independent of the route of vaccine application, viremia was significantly reduced in vaccinees compared with controls 2 weeks post‐challenge. Concomitantly, viremia correlated inversely with SIV‐specific IgG, complement‐mediated lysis and neutralizing antibodies and these parameters seemed to contribute to reduced viremia. During chronic infection, six monkeys controlled viremia in the circulation (two or fewer infectious units per 10⁶ PBMCs) and showed no signs of trapping in lymphatic tissues (Appendix S1). Conclusions As no significant differences were observed throughout the study, with respect to the humoral immune response and viremia control, between the two vaccinated cohorts, mucosal immunization strategies are recommended due to more simplified application.     

Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV

Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.     

Resistance to Infection,Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251 Viremia,and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIV_mac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIV_mac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8⁺ T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4⁺ central memory and CD4⁺/α4β7⁺ T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4⁺, and CD8⁺ T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.     

Immunogenicity of Seven New Recombinant Yellow Fever Viruses 17D Expressing Fragments of SIVmac239 Gag,Nef, and Vif in Indian Rhesus Macaques

An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8⁺ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4⁺ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D – a clinically relevant vaccine vector – can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.     

Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T-cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4⁺ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4⁺ T-cell play roles in protection against wild-type virus challenge.
Methods  Rhesus macaques were vaccinated with SIVmac239Δnef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies.
Results  Animals vaccinated with SIVmac239Δnef demonstrated no loss of CD4⁺ T cells in any tissue, and in fact CCR5⁺ and CD28⁺CD95⁺ central memory CD4⁺ T cells were significantly increased. In contrast, CD4⁺ T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Δnef vaccination then declined following primary infection.
Conclusion  We demonstrated in this study that SIVmac239Δnef vaccination did not deplete CD4⁺ T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4⁺ T cells did not appear to influence protective immunity.     

Protection of Rabbits from Viral Challenge by Gene Gun-Based Intracutaneous Vaccination with a Combination of Cottontail Rabbit Papillomavirus E1, E2, E6, and E7 Genes

In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.     

The immunopathogenesis of retroviral diseases: No immunophenotypic alterations in T,B, and NK cell subsets in SIVmac239-challenged rhesus macaques protected by SIVΔnef vaccination

Abstract: Immunophenotype analysis was used to characterize circulating lymphocyte subset levels in both rhesus monkeys that were chronically infected with SIV_mac239 and in those that had resisted SIV_mac239 infection as a result of prior vaccination with an attenuated SIV strain. Alterations in T, NK, and B cell subsets were compared with those previously identified in humans chronically infected with HIV [8–11, 14, 22]. The well-known decrease in CD4⁺ cell levels was observed in the SIV_mac239-infected animals. However, these animals had relatively little activation of circulating CD8⁺ T cells as compared with uninfected monkeys. This contrasts with chronically HIV-infected humans who have substantial activation of circulating CD8⁺ cells as evidenced by elevated HLA-DR and CD38 antigen expression on CD8⁺ cells as well as substantially increased percentages and numbers of total CD8⁺ cells. NK cells of the SIV_mac239-infected animals, on the other hand, demonstrated the same changes recently described in HIV-infected humans, i.e., a decrease in circulating percentages and a decreased amount of FcRIII (CD 16). B cell percentages were markedly increased in the SIV_mac239-infected animals, a finding also noted in some children with HIV infection but not in HIV-infected adults. SIVΔnef-vaccinated/SIV_mac239-challenged animals showed none of the immune alterations found in the SIV_mac239-infected monkeys, providing further confirmation of lack of SIV disease in these vaccinated animals.     

Retroviral recombination in vivo: viral replication patterns and genetic structure of simian immunodeficiency virus (SIV) populations in rhesus macaques after simultaneous or sequential intravaginal inoculation with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef

To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Deltavpx/Deltavpr and then with SIVmac239Deltanef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Deltanef when plasma SIVmac239Deltavpx/Deltavpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Deltanef, and then with SIVmac239Deltavpx/Deltavpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.     

Characterization of GPI2A,a Potent Inhibitor of HIV-1 Gene Expression and Viral Replication

Abstract

Fully thioated antisense molecules are often cytotoxic and non-specitic in action. GPI2A is thioated at 7 base positions. GPI2A posses sequence-specific activity against HIV-1 gene expression and viral replication without sigdcant cytotoxicity. Partial thioation did not compromise its uptake, cellular distribution and nuclease resistance.     

伪狂犬病毒gI基因的克隆表达及其对病毒增殖的影响

从伪狂犬病毒(PRV)国内地方分离Ea株基因组DNA片段中克隆了完整的gI基因,序列分析结果表明,gI基因编码区全长1101bp,可编码366个氨基酸残基,二级结构预测具有典型I型膜蛋白特征。与GenBank中收录的国外Rice株的同源比较发现,Ea株gI在核苷酸和氨基酸水平上均存在多处突变,尤其是潜在胞浆区中连续两个碱基的缺失导致移码突变,致使gI基因的读码框架后移,从而导致Ea株gI较rice株长16个氨基酸残基。将gI基因克隆到真核表达载体pcDNA31+中的人巨细胞病毒早期启动子下游,构建的真核表达质粒转染PK15细胞,间接免疫荧光检测证实gI获得正确表达。进一步测定天然缺失gI的PRV弱毒Bartha株在表达gI细胞系和空白载体转染的对照细胞系中的蚀斑形成单位(pfu)和组织细胞培养半数感染量(TCID50),结果显示：Bartha株在表达gI细胞系中的pfu和TCID\-\{50\}分别为对照细胞系的164%和200%。说明gI具有促进病毒增殖的功能。     

Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.     

Immunization of macaques with single-cycle simian immunodeficiency virus (SIV) stimulates diverse virus-specific immune responses and reduces viral loads after challenge with SIVmac239

Genetically engineered simian immunodeficiency viruses (SIV) that is limited to a single cycle of infection was evaluated as a nonreplicating AIDS vaccine approach for rhesus macaques. Four Mamu-A*01(+) macaques were inoculated intravenously with three concentrated doses of single-cycle SIV (scSIV). Each dose consisted of a mixture of approximately equivalent amounts of scSIV strains expressing the SIV(mac)239 and SIV(mac)316 envelope glycoproteins with mutations in nef that prevent major histocompatibility complex (MHC) class I downregulation. Viral loads in plasma peaked between 10(4) and 10(5) RNA copies/ml on day 4 after the first inoculation and then steadily declined to undetectable levels over the next 4 weeks. SIV Gag-specific T-cell responses were detected in peripheral blood by MHC class I tetramer staining (peak, 0.07 to 0.2% CD8(+) T cells at week 2) and gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assays (peak, 50 to 250 spot forming cells/10(6) peripheral blood mononuclear cell at week 3). Following the second and third inoculations at weeks 8 and 33, respectively, viral loads in plasma peaked between 10(2) and 10(4) RNA copies/ml on day 2 and were cleared over a 1-week period. T-cell-proliferative responses and antibodies to SIV were also observed after the second inoculation. Six weeks after the third dose, each animal was challenged intravenously with SIV(mac)239. All four animals became infected. However, three of the four scSIV-immunized animals exhibited 1 to 3 log reductions in acute-phase plasma viral loads relative to two Mamu-A*01(+) control animals. Additionally, two of these animals were able to contain their viral loads below 2,000 RNA copies/ml as late as 35 weeks into the chronic phase of infection. Given the extraordinary difficulty in protecting against SIV(mac)239, these results are encouraging and support further evaluation of lentiviruses that are limited to a single cycle of infection as a preclinical AIDS vaccine approach.     

Infectivity and Immunogenicity of SIVmac/HIV-1 Chimeric Viruses (SHIVs) with Deletions in Two or Three Genes (vpr,nef and vpx)

Two SHIVs with two or three genes deleted (SHIV-drn and SHIV-dxrn) were constructed. The inoculation of monkeys with SHIV-drn resulted in short-term viremia, but inoculation with SHIV-dxrn did not. At 68 weeks post-inoculation, the monkeys were reinoculated with a 100-fold higher dose of each SHIV, but none showed viremia. Killer cell activities against HTV-1 Env were detected in the SHIV-drn- and SHIV-dxrn-inoculated monkeys. Cross-reactive killer activity against HIV-1 Gag and SIVmac Gag was observed in one monkey. Antibodies were not detected in the SHIV-dxrn-inoculated monkeys, but the SHIV-drn-inoculated monkeys showed an anamnestic antibody reaction. These data indicate that SHIV-drn is infectious to and immuno-inducible in macaques but SHIV-dxrn is not.     

Contributions of Mamu-A*01 Status and TRIM5 Allele Expression,But Not CCL3L Copy Number Variation,to the Control of SIVmac251 Replication in Indian-Origin Rhesus Monkeys

CCL3 is a ligand for the HIV-1 co-receptor CCR5. There have recently been conflicting reports in the literature concerning whether CCL3-like gene (CCL3L) copy number variation (CNV) is associated with resistance to HIV-1 acquisition and with both viral load and disease progression following infection with HIV-1. An association has also been reported between CCL3L CNV and clinical sequelae of the simian immunodeficiency virus (SIV) infection in vivo in rhesus monkeys. The present study was initiated to explore the possibility of an association of CCL3L CNV with the control of virus replication and AIDS progression in a carefully defined cohort of SIVmac251-infected, Indian-origin rhesus monkeys. Although we demonstrated extensive variation in copy number of CCL3L in this cohort of monkeys, CCL3L CNV was not significantly associated with either peak or set-point plasma SIV RNA levels in these monkeys when MHC class I allele Mamu-A*01 was included in the models or progression to AIDS in these monkeys. With 66 monkeys in the study, there was adequate power for these tests if the correlation of CCL3L and either peak or set-point plasma SIV RNA levels was 0.34 or 0.36, respectively. These findings call into question the premise that CCL3L CNV is important in HIV/SIV pathogenesis.     

Low-dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the phase IIb step trial of a similar HIV-1 vaccine

The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.     

Potent,persistent induction and modulation of cellular immune responses in rhesus macaques primed with Ad5hr-simian immunodeficiency virus (SIV) env/rev,gag, and/or nef vaccines and boosted with SIV gp120

Immunity elicited by multicomponent vaccines delivered by replication-competent Ad5hr-simian immunodeficiency virus (SIV) recombinants was systematically investigated. Rhesus macaques were immunized mucosally at weeks 0 and 12 with Ad5hr-SIV(smH4) env/rev, with or without Ad5hr-SIV(mac239) gag or Ad5hr-SIV(mac239) nef, or with all three recombinants. The total Ad5hr dosage was comparably adjusted among all animals with empty Ad5hr-DeltaE3 vector. The macaques were boosted with SIV gp120 in monophosphoryl A-stable emulsion adjuvant at 24 and 36 weeks. Controls received Ad5hr-DeltaE3 vector or adjuvant only. By ELISPOT analysis, all four SIV gene products elicited potent cellular immune responses that persisted 42 weeks post-initial immunization. Unexpectedly, modulation of this cellular immune response was observed among macaques receiving one, two, or three Ad5hr-SIV recombinants. Env responses were significantly enhanced throughout the immunization period in macaques immunized with Ad5hr-SIV env/rev plus Ad5hr-SIV gag and tended to be higher in macaques that also received Ad5hr-SIV nef. Macaques primed with all three recombinants displayed significant down-modulation in numbers of gamma interferon (IFN-gamma)-secreting cells specific for SIV Nef, and the Env- and Gag-specific responses were also diminished. Modulation of antibody responses was not observed. Down-modulation was seen only during the period of Ad5hr-recombinant priming, not during subunit boosting, although SIV-specific IFN-gamma-secreting cells persisted. The effect was not attributable to Ad5hr replication differences among immunization groups. Vaccine delivery via replication-competent live vectors, which can persistently infect new cells and continuously present low-level antigen, may be advantageous in overcoming competition among complex immunogens for immune recognition. Effects of current multicomponent vaccines on individual immune responses should be evaluated with regard to future vaccine design.