SIV Mac251 Research Articles

Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy.

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIV mac 239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

IL-21 enhances influenza vaccine responses in aged macaques with suppressed SIV infection.

Natural aging and HIV infection are associated with chronic low-grade systemic inflammation, immune senescence, and impaired antibody responses to vaccines such as the influenza (flu) vaccine. We investigated the role of IL-21, a CD4+ T follicular helper cell (Tfh) regulator, on flu vaccine antibody response in nonhuman primates (NHPs) in the context of age and controlled SIV mac239 infection. Three doses of the flu vaccine with or without IL-21–IgFc were administered at 3-month intervals in aged SIV+ NHPs following virus suppression with antiretroviral therapy. IL-21–treated animals demonstrated higher day 14–postboost antibody responses, which associated with expanded CD4+ T central memory cells and peripheral Tfh–expressing (pTfh–expressing) T cell immunoreceptor with Ig and ITIM domains (TIGIT), expanded activated memory B cells, and contracted CD11b+ monocytes. Draining lymph node (LN) tissue from IL-21–treated animals revealed direct association between LN follicle Tfh density and frequency of circulating TIGIT+ pTfh cells. We conclude that IL-21 enhances flu vaccine–induced antibody responses in SIV+ aged rhesus macaques (RMs), acting as an adjuvant modulating LN germinal center activity. A strategy to supplement IL-21 in aging could be a valuable addition in the toolbox for improving vaccine responses in an aging HIV+ population.

The autophagy protein ATG9A promotes HIV-1 infectivity

BackgroundNef is a multifunctional accessory protein encoded by HIV-1, HIV-2 and SIV that plays critical roles in viral pathogenesis, contributing to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways.ResultsTo gain a better understanding of the role of host proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV (cpz, smm and mac). However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we knocked out ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A knockout (KO) had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by ~ fourfold and eightfold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein.ConclusionsThese studies indicate that ATG9A promotes HIV-1 infectivity in an Env-dependent manner. The interaction of Nef with ATG9A, however, is not required for Nef to enhance HIV-1 infectivity. We speculate that ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. These studies thus identify a novel host cell factor implicated in HIV-1 infectivity, which may be amenable to pharmacologic manipulation for treatment of HIV-1 infection.

Effect of morphine and SIV on dendritic cell trafficking into the central nervous system of rhesus macaques

Recruitment of immune cells such as monocytes/macrophages and dendritic cells (DCs) across the blood-brain barrier (BBB) has been documented in diseases involving neuroinflammation. Neuroinvasion by HIV leads to neurocognitive diseases and alters the permeability of the BBB. Likewise, many HIV patients use drugs of abuse such as morphine, which can further compromise the BBB. While the role of monocytes and macrophages in neuroAIDS is well established, research demonstrating the presence and role of DCs in the CNS during HIV infection has not been developed yet. In this respect, this study explored the presence of DCs in the brain parenchyma of rhesus macaques infected with a neurovirulent form of SIV (SIV mac239 R71/17E) and administered with morphine. Cells positive for DC markers including CD11c (integrin), macDC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), CD83 (a maturation factor), and HLA-DR (MHC class II) were consistently found in the brain parenchyma of SIV-infected macaques as well as infected macaques on morphine. Control animals did not exhibit any DC presence in their brains. These results provide first evidence of DCs' relevance in NeuroAIDS vis-à-vis drugs of abuse and open new avenues of understanding and investigative HIV-CNS inflictions.

Protection afforded by an HIV vaccine candidate in macaques depends on the dose of SIVmac251 at challenge exposure.

We used the simian immunodeficiency virus mac251 (SIV(mac251)) macaque model to study the effect of the dose of mucosal exposure on vaccine efficacy. We immunized macaques with a DNA prime followed by SIV gp120 protein immunization with ALVAC-SIV and gp120 in alum, and we challenged them with SIV(mac251) at either a single high dose or at two repeated low-dose exposures to a 10-fold-lower dose. Infection was neither prevented nor modified following a single high-dose challenge of the immunized macaques. However, two exposures to a 10-fold-lower dose resulted in protection from SIV(mac251) acquisition in 3 out of 12 macaques. The remaining animals that were infected had a modulated pathogenesis, significant downregulation of interferon responsive genes, and upregulation of genes involved in B- and T-cell responses. Thus, the choice of the experimental model greatly influences the vaccine efficacy of vaccines for human immunodeficiency virus (HIV).

Evidence for Env-V2 sieve effect in breakthrough SIV MAC251 infections in rhesus monkeys vaccinated with Ad26/MVA and MVA/Ad26 constructs

Results The mean pairwise AA diversity among the 66 SIVMAC251 Env sequences was 0.38%, and they differed from the vaccine strain SIV SME543 (Env) by 21.94%. The repeated low-dose challenge resulted in infections with an average of 1.7 founder variants with no evidence that the vaccine restricted the number of variants (p = 0.813). We explored whether the vaccine induced a sieve effect, i.e. whether breakthrough viruses differed between the vaccine and control groups. There was no difference for full-length Env sequences. Focusing on Env segments preferentially recognized by vaccinated monkeys in antibody arrays, we identified a sieve effect in the Env-V2 segment AA163-193: sequences from vaccinated animals were more divergent from the vaccine SIVSME543 or from the challenge stock SIVMAC251 than sequences in control animals (p ≤ 0.002).

Immune complexes can dampen inflammatory signaling at the mucosal surface during protective SIV vaccination

Background The long-term goal for an efficacious HIV vaccine is to provide sterilizing protection from HIV infection. Thus far, this scenario has only been achieved experimentally using live-attenuated SIV vaccines. As such, great interest lies in identifying correlates of protection from a successful host response to pathogenic SIV. To this end, we have used a global genomics approach, tissue analysis, and explant cultures to identify immune complex (IC) signaling as an important component of a protective host response in the female reproductive tract (FRT) of animals vaccinated with the live-attenuated virus known as SIV mac239 ΔNef.

Immunization with apoptotic pseudovirus transduced cells induces both cellular and humoral responses: A proof of concept study in macaques

Dendritic cells are able to present viral antigens to T-cells after uptake of apoptotic bodies derived from virus-infected cells. Immunization with virus-infected apoptotic cells was previously shown to induce HIV-specific immune responses in mice. Here we evaluate the safety and immunogenicity of immunization with activated apoptotic cells in non-human primates using autologous T-cells infected with replication defective VSV pseudotyped SIV(mac239)Δenv. Animals were immunized with γ-irradiated activated T-cells carrying the VSVenvSIV(mac239)Δenv pseudovirus. SIV Gag-specific cellular immune responses were induced as early as two weeks after the first immunization eliciting a biased IFN-γ and IL-2 response. In addition, induction of SIV Gag-specific antibody responses and high titer neutralizing activity against the SIV pseudovirus harboring a VSV-env were detected after two immunizations. The vaccinated group and a control group of Chinese rhesus macaques were intravenously challenged with pathogenic SIV(mac251.) All animals became infected, but SIV-replication was effectively suppressed (below 100 copies/ml) in several animals in both groups. However the group immunized with apoptotic cells revealed better preservation of the gut CD4(+) T-cell compartment. Viral control was inversely correlated with an early (4 weeks) but transient increase in the percentage of Ki67(+)CD4(+) peripheral blood T-cells (Spearman -0.73). We here show that immunizations with activated apoptotic lymphocytes expressing transduced SIV genes result in induction of both cellular and humoral immune responses. This study provides evidence for an immunological principle demonstrating that certain apoptotic cells can be considered as carriers of antigens directing immune responses in macaques.

The role of MHC class I allele Mamu-A*07 during SIV(mac)239 infection.

Virus-specific CD8(+) T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n = 63 of 1,240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIV(mac)239 for their capacity to bind Mamu-A1*007:01; 33 were found to bind with an affinity of 500nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T-cell reactivity. In all, 11 of the peptides elicited IFN-γ(+) T-cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultradeep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8(+) T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8(+) T cells and is part of the immune response to SIV(mac)239.

A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5′ untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIVMac coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role.

Rabies virus-based vaccines elicit neutralizing antibodies, poly-functional CD8 + T cell, and protect rhesus macaques from AIDS-like disease after SIV mac251 challenge

Highly attenuated rabies virus (RV) vaccine vectors were evaluated for their ability to protect against highly pathogenic SIV mac251 challenge. Mamu-A*01 negative rhesus macaques were immunized in groups of four with either: RV expressing SIV mac239-GagPol, a combination of RV expressing SIV mac239-Env and RV expressing SIV mac239-GagPol, or with empty RV vectors. Eight weeks later animals received a booster immunization with a heterologous RV expressing the same antigens. At 12 weeks post-boost, all animals were challenged intravenously with 100 TCID 50 of pathogenic SIV mac251-CX. Immunized macaques in both vaccine groups had 1.3–1.6-log-fold decrease in viral set point compared to control animals. The GagPol/Env immunized animals also had a significantly lower peak viral load. When compared to control animals following challenge, vaccinated macaques had a more rapid induction of SIV mac251 neutralizing antibodies and of CD8 + T cell responses to various SIV epitopes. Moreover, vaccinated macaques better maintained peripheral memory CD4 + T cells and were able to mount a poly-functional CD8 + T cell response in the mucosa. These findings indicate promise for RV-based vectors and have important implications for the development of an efficacious HIV vaccine.

Debio-025 inhibits HIV-1 by interfering with an early event in the replication cycle

Cyclophilin A is a peptidyl-propyl isomerase that binds the capsid (p24) protein of HIV-1 and facilitates replication. We report a cyclophilin inhibitor, a non-immunosuppressive cyclosporine analogue, Debio-025, that is about 15-times more potent than cyclosporine A and less toxic resulting in a selectivity index of more than 300. It was equally active against virus strains that were resistant toward inhibitors of the viral entry, fusion, or reverse transcription while it was not inhibitory to HIV-2 or SIV MAC. Mechanism of action studies demonstrate that Debio-025 inhibits the HIV-1 replication by interfering with an early stage of the viral replication cycle. Indeed, addition of Debio-025 could be postponed for 2 h before loosing its antiviral activity. The compound proved inactive against mutant viruses that are independent of cyclophilin A binding suggesting Debio-025 targets the cyclophilin A–capsid interaction.

The CDK inhibitor p21Cip1/WAF1 is induced by FcgammaR activation and restricts the replication of human immunodeficiency virus type 1 and related primate lentiviruses in human macrophages.

Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1). We have previously shown that aggregation of activating immunoglobulin G Fc receptors (FcgammaR) by immune complexes inhibits reverse transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages. Here, we show that FcgammaR-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) (p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in FcgammaR-activated macrophages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macrophages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral replication independently of a specific interaction with an HIV-1 component. Consistently, p21 silencing rescued viral replication from the FcgammaR-mediated restriction also in simian immunodeficiency virus SIV(mac)- and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral infection in macrophages and to its implication in FcgammaR-mediated restriction.

Nef-mediated MHC class I down-regulation unmasks clonal differences in virus suppression by SIV-specific CD8(+) T cells independent of IFN-gamma and CD107a responses.

CD8(+) T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8(+) T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4(+) T cells. We used a set of SIV(mac)239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I(high) or MHC class I(intermediate). However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I(low)) despite the observations that all CTL clones showed similar IFN-gamma responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.

Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIVmac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIVsmm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.

Control of viremia and maintenance of intestinal CD4 + memory T cells in SHIV 162P3 infected macaques after pathogenic SIV MAC251 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.

The mechanism involved in the repression of the μ opioid receptor gene expression in CEM ×174 cells infected by simian immunodeficiency virus

Morphine can promote the pathogenesis of human acquired immunodeficiency syndrome through binding to the mu opioid receptor (MOR) in immune cells. Previous investigation has suggested that expression of the MOR gene in lymphocytes is triggered by cooperative interaction between transcription factors, specificity protein 1 (Sp1) and Ying Yang 1 (YY1), in the promoter region. However, the specific molecular mechanism by which immunodeficiency virus infection impacts regulation of the MOR gene expression in lymphocytes is still unclear. In this study, it was demonstrated that SIV (SIVmac239) infection may result in gradual reduction of the MOR gene expression and Sp1 during a period of 48 h postinfection by analysis of quantitative real-time RT-PCR and Western blotting. The results of methylation-specific PCR showed that two of 14 CpG islands adjacent to the Sp1 and YY1 elements in the promoter region were methylated, which together with reduced Sp1, contributed to the failure of interaction of Sp1 with YY1 and their binding to the elements, as determined by coimmunoprecipitation, chromatin immunoprecipitation-real-time PCR, and EMSAs. The repression of the MOR gene secondary to SIVmac239 infection could be abolished by the demethylating agent 5-aza-2'-deoxycytidine. Transfection with Sp1-expressing vector (PN3-Sp1) was also able to enhance the activity of the promoter in SIVmac239-infected cells. We therefore concluded that aberrant methylation of the promoter and reduction of Sp1 resulting from SIVmac239 infection led to the silencing of the MOR gene. This finding will be helpful in understanding the synergistic mechanism of HIV infection and morphine addiction in the pathogenesis of AIDS.

The Mamu B ⁎17-restricted SIV Nef IW9 to TW9 mutation abrogates correct epitope processing and presentation without loss of replicative fitness

CD8 + cytotoxic T lymphocytes (CTL) play an important role in controlling virus replication in HIV- and SIV-infected humans and monkeys, respectively. Three well-studied SIV CTL determinants are the two Mamu A ⁎01-restricted epitopes Gag CM9 and Tat SL8, and the Mamu B ⁎17-restricted epitope Nef IW9. Point mutations leading to amino acid replacements in these epitopes have been reported to mediate SIV escape from CTL control. We found that synthetic peptides containing mutations in SIV Gag CM9 and Tat SL8 were no longer recognized by the respective CTL. On the other hand, the described I-to-T replacement at the N-terminal amino acid residue of the SIV Nef IW9 epitope only moderately affected CTL recognition of the variant peptide, TW9. In an attempt to dissect the mechanism of escape of the Nef TW9 mutation, we investigated the effect of this mutation on CTL recognition of CD4 +T cells infected with an engineered SIV mac239 that contained the TW9 mutation in Nef. Although, the wild type and mutant virus both infected and efficiently replicated in rhesus macaque CD4 +T cells, the TW9 mutant virus failed to induce IFN-γ expression in an SIV Nef IW9-specific CTL clone. Thus, unlike escape from Gag CM9- or Tat SL8-specfic CTL control presumably by loss of epitope binding, these results point to a defect at the level of processing and/or presentation of the variant TW9 epitope with resultant loss of triggering of the cognate TCR on CTL generated against the wild type peptide. Our data highlight the value of functional assays using virus-infected target cells as opposed to peptide-pulsed APC when assessing relevant escape mutations in CTL epitopes.

Efficient inhibition of SIV replication in rhesus CD4 + T-cell clones by autologous immortalized SIV-specific CD8 + T-cell clones

CD8 + cytotoxic T lymphocyte (CTL) responses play an important role in controlling the replication of primate lentiviruses. Induction of these responses is a key objective for most current AIDS vaccine approaches. Despite a variety of approaches for measuring properties and activities of CTL, the functions responsible for controlling viral replication in vivo have not been clearly identified. Assays measuring CTL-mediated suppression of viral replication in vitro are beginning to be used as possible correlates of in vivo virus suppressive activity, but the utility and interpretive value of these assays are typically limited by properties of the cells that have been used. We investigated the capacity of SIV-specific CTL clones (effectors), immortalized by transduction with human telomerase reverse transcriptase (hTERT), to suppress SIV replication in autologous hTERT immortalized CD4 + T-cell clones (targets). Immortalized and non-immortalized SIV-specific effector cells showed IFN-γ production and degranulation in response to viral antigen specific stimulation and significantly inhibited SIV mac239 replication (2 to 4 log decrease in viral RNA or cell-associated proviral DNA) ( p < 0.0005). Our in vitro assays of inhibition of viral replication, using T-cell clones as effectors and targets, provide a well-defined approach for evaluating possible mechanisms of CTL-mediated control of viral production which may involve direct killing of infected target cells and/or release of proinflammatory cytokines such as IFN-γ and TNF-α. The use of hTERT immortalized effector and target cells for such assays preserves relevant functional properties while providing a convenient, reproducible means of conducting studies over time.

Evaluation of recombinant Kunjin replicon SIV vaccines for protective efficacy in macaques

Persistent gag-specific T cell immunity would be a useful component of an effective HIV vaccine. The Flavivirus Kunjin replicon was previously engineered to persistently express HIV gag and was shown to induce protective responses in mice. We evaluated Kunjin replicon virus-like-particles expressing SIVgag–pol in pigtail macaques. Kunjin-specific antibodies were induced, but no SIV-specific T cell immunity were detected. Following SIV mac251 challenge, there was no difference in SIV viremia or retention of CD4 T cells between Kunjin–SIVgag–pol vaccine immunized animals and controls. An amnestic SIV gag-specific CD8 T cell response associated with control of viremia was observed in 1 of 6 immunized animals. Refinements of this vector system and optimization of the immunization doses, routes, and schedules are required prior to clinical trials.