Search

Macaque recipients of anti–HIV-1 combination NAb treatment starting on PI day 3 or week 2 generate CD8+ cellular immunity that maintains durable suppression of SHIV replication and circulating CD4+ T cell levels during a 3–6-yr observation period., We report that combination bNAb immunotherapy initiated on day 3 post-infection (PI) maintained durable CD8+ T cell–mediated suppression of SHIVAD8 viremia and preinoculation levels of CD4+ T cells in 9 of 13 treated monkeys during nearly 6 yr of observation, as assessed by successive CD8+ T cell–depletion experiments. In an extension of that study, two treatment interventions (bNAbs alone or cART plus bNAbs) beginning on week 2 PI were conducted and conferred controller status to 7 of 12 monkeys that was also dependent on control mediated by CD8+ cells. However, the median time to suppression of plasma viremia following intervention on week 2 was markedly delayed (85 wk) compared with combination bNAb immunotherapy initiated on day 3 (39 wk). In both cases, the principal correlate of virus control was the induction of CD8+ T cellular immunity.

In the absence of an effective and safe vaccine against HIV-1, the administration of broadly neutralizing antibodies (bNAbs) represents a logical alternative approach to prevent virus transmission. Here, we introduced two mutations encoding amino acid substitutions (M428L and N434S, collectively referred to as ‘LS’) into the genes encoding the crystallizable fragment domains of the highly potent HIV-specific 3BNC117 and 10-1074 bNAbs to increase their half-lives and evaluated their efficacy in blocking infection following repeated low-dose mucosal challenges of rhesus macaques (Macaca mulatta) with the tier 2 SHIVAD8-EO. A single intravenous infusion of 10-1074-LS monoclonal antibodies markedly delayed virus acquisition for 18 to 37 weeks (median, 27 weeks), whereas the protective effect of the 3BNC117-LS bNAb was more modest (provided protection for 11–23 weeks; median, 17 weeks). Serum concentrations of the 10-1074-LS monoclonal antibody gradually declined and became undetectable in all recipients between weeks 26 and 41, whereas the 3BNC117-LS bNAb exhibited a shorter half-life. To model immunoprophylaxis against genetically diverse and/or neutralization-resistant HIV-1 strains, a combination of the 3BNC117-LS plus 10-1074-LS monoclonal antibodies was injected into macaques via the more clinically relevant subcutaneous route. Even though the administered mixture contained an amount of each bNAb that was nearly threefold less than the quantity of the single monoclonal antibody in the intravenous injections, the monoclonal antibody combination still protected macaques for a median of 20 weeks. The extended period of protection observed in macaques for the 3BNC117-LS plus 10-1074-LS combination could translate into an effective semiannual or annual immunoprophylaxis regimen for preventing HIV-1 infections in humans.

APOBEC3G (A3G) is a cytidine deaminase with potent antiviral activity that is antagonized by Vif. A3G is expressed in a cell type-specific manner and some semi-permissive cells, including A3.01, express A3G but fail to block replication of Vif-null HIV-1. Here we explored the semi-permissive nature of A3.01 cells and found it to be defined exclusively by the levels of A3G. Indeed, minor changes in A3G levels rendered A3.01 cells either fully permissive or non-permissive for Vif-null HIV-1. Our data indicate that A3.01 cells express sub-lethal levels of catalytically active A3G that affects Vif-null HIV-1 at the proviral level but does not completely block virus replication due to purifying selection. Attempts to use the selective pressure exerted by such sub-lethal levels of A3G to select for APOBEC-resistant Vif-null virus capable of replicating in H9 cells failed despite passaging virus for five months, demonstrating that Vif is a critical viral accessory protein.

TRIM5α polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of human immunodeficiency virus (HIV) vaccine strategies in rhesus macaques. We previously reported that the TRIM5α-sensitive SIV from sooty mangabeys (SIVsm) clone SIVsmE543-3 acquired amino acid substitutions in the capsid that overcame TRIM5α restriction when it was passaged in rhesus macaques expressing restrictive TRIM5α alleles. Here we generated TRIM5α-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5α alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5α genotypes as efficiently as in macaques with permissive TRIM5α genotypes. These results demonstrated that mutations in the SIV capsid can confer SIV resistance to TRIM5α restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies. IMPORTANCE Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5α alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques, and the introduction of two amino acid changes in the capsid transfers this improved replication to the parent clone. In the present study, we introduced these mutations into a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved the replication of this strain in macaques with the restrictive TRIM5α genotype and thus will eliminate the confounding effects of TRIM5α in vaccine studies.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 . Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions. IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus . Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.

Nonhuman primate-simian immunodeficiency virus (SIV) models are powerful tools for studying the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in the brain. Our laboratory recently isolated a neuropathogenic viral swarm, SIVsmH804E, a derivative of SIVsmE543-3, which was the result of sequential intravenous passages of viruses isolated from the brains of rhesus macaques with SIV encephalitis. Animals infected with SIVsmH804E or its precursor (SIVsmH783Br) developed SIV meningitis and/or encephalitis at high frequencies. Since we observed macaques with a combination of meningitis and encephalitis, as well as animals in which meningitis or encephalitis was the dominant component, we hypothesized that distinct mechanisms could be driving the two pathological states. Therefore, we assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection. Viral RNAs were isolated from representative areas of the meninges, brain parenchyma, terminal plasma, and cerebrospinal fluid (CSF) and from the inoculum, and the SIV envelope fragment was amplified by PCR. Phylogenetic analysis of envelope sequences from the conventional progressors revealed compartmentalization of viral populations between the meninges and the parenchyma. In one of these animals, viral populations in meninges were closely related to those from CSF and shared signature truncations in the cytoplasmic domain of gp41, consistent with a common origin. Apart from magnetic resonance imaging (MRI) and positron-emission tomography (PET) imaging, CSF is the most accessible assess to the central nervous system for HIV-1-infected patients. However, our results suggest that the virus in the CSF may not always be representative of viral populations in the brain and that caution should be applied in extrapolating between the properties of viruses in these two compartments.

Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.

Early administration of broadly neutralizing antibodies in a macaque SHIV infection model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant long-term infection control. No effective vaccine has yet been developed against the human immunodeficiency virus type 1 (HIV-1), which is endemic in many areas of the world. Yoshiaki Nishimura et al. studied the effects of giving broadly neutralizing antibodies against HIV-1 to rhesus macaques shortly after beginning of exposure to low doses of the simian/human immunodeficiency virus (SHIV). This type of immunotherapy was associated with the persistence of low levels of the virus in the animals' blood, which led to the establishment of T-cell immunity and resulted in enhanced infection control. These findings could stimulate research into the mechanisms of HIV-1 control in infected humans. Highly potent and broadly neutralizing anti-HIV-1 antibodies (bNAbs) have been used to prevent and treat lentivirus infections in humanized mice, macaques, and humans1,2,3,4,5,6,7,8,9,10,11,12. In immunotherapy experiments, administration of bNAbs to chronically infected animals transiently suppresses virus replication, which invariably returns to pre-treatment levels and results in progression to clinical disease. Here we show that early administration of bNAbs in a macaque simian/human immunodeficiency virus (SHIV) model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant long-term infection control. Animals challenged with SHIVAD8-EO by mucosal or intravenous routes received a single 2-week course of two potent passively transferred bNAbs (3BNC117 and 10-1074 (refs 13, 14)). Viraemia remained undetectable for 56–177 days, depending on bNAb half-life in vivo. Moreover, in the 13 treated monkeys, plasma virus loads subsequently declined to undetectable levels in 6 controller macaques. Four additional animals maintained their counts of T cells carrying the CD4 antigen (CD4+) and very low levels of viraemia persisted for over 2 years. The frequency of cells carrying replication-competent virus was less than 1 per 106 circulating CD4+ T cells in the six controller macaques. Infusion of a T-cell-depleting anti-CD8β monoclonal antibody to the controller animals led to a specific decline in levels of CD8+ T cells and the rapid reappearance of plasma viraemia. In contrast, macaques treated for 15 weeks with combination anti-retroviral therapy, beginning on day 3 after infection, experienced sustained rebound plasma viraemia when treatment was interrupted. Our results show that passive immunotherapy during acute SHIV infection differs from combination anti-retroviral therapy in that it facilitates the emergence of potent CD8+ T-cell immunity able to durably suppress virus replication.

A single injection of four anti-HIV-1-neutralizing monoclonal antibodies blocks repeated weekly low-dose virus challenges of simian/human immunodeficiency virus. This study assesses the long-term efficacy of a passive antibody transfer approach for the control of human immunodeficiency virus type 1 (HIV-1) infection. Malcolm Martin and colleagues administered single intravenous injections of four different anti-HIV-1 neutralizing monoclonal antibodies in a simian/HIV intrarectal exposure model involving weekly low-dose viral challenge and demonstrate protection from infection lasting almost 6 months. Despite the success of potent anti-retroviral drugs in controlling human immunodeficiency virus type 1 (HIV-1) infection, little progress has been made in generating an effective HIV-1 vaccine. Although passive transfer of anti-HIV-1 broadly neutralizing antibodies can protect mice or macaques against a single high-dose challenge with HIV or simian/human (SIV/HIV) chimaeric viruses (SHIVs) respectively1,2,3,4,5,6,7,8, the long-term efficacy of a passive antibody transfer approach for HIV-1 has not been examined. Here we show, on the basis of the relatively long-term protection conferred by hepatitis A immune globulin, the efficacy of a single injection (20 mg kg−1) of four anti-HIV-1-neutralizing monoclonal antibodies (VRC01, VRC01-LS, 3BNC117, and 10-1074 (refs 9, 10, 11, 12)) in blocking repeated weekly low-dose virus challenges of the clade B SHIVAD8. Compared with control animals, which required two to six challenges (median = 3) for infection, a single broadly neutralizing antibody infusion prevented virus acquisition for up to 23 weekly challenges. This effect depended on antibody potency and half-life. The highest levels of plasma-neutralizing activity and, correspondingly, the longest protection were found in monkeys administered the more potent antibodies 3BNC117 and 10-1074 (median = 13 and 12.5 weeks, respectively). VRC01, which showed lower plasma-neutralizing activity, protected for a shorter time (median = 8 weeks). The introduction of a mutation that extends antibody half-life into the crystallizable fragment (Fc) domain of VRC01 increased median protection from 8 to 14.5 weeks. If administered to populations at high risk of HIV-1 transmission, such an immunoprophylaxis regimen could have a major impact on virus transmission.

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4 + T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.

There is an urgent need to develop new pathogenic R5 simian/human immunodeficiency viruses (SHIVs) for the evaluation of candidate anti-HIV vaccines in nonhuman primates. Here, we characterize swarm SHIV AD8 stocks, prepared from three infected rhesus macaques with documented immunodeficiency at the time of euthanasia, for their capacity to establish durable infections in macaques following inoculation by the intravenous (i.v.) or intrarectal (i.r.) route. All three viral stocks (SHIV AD8-CE8J , SHIV AD8-CK15 , and SHIV AD8-CL98 ) exhibited robust replication in vivo and caused marked depletion of CD4 + T cells affecting both memory and naïve CD4 + T lymphocyte subsets following administration by either route. Eleven of 22 macaques inoculated with the new SHIV AD8 stocks were euthanized with clinical symptoms of immunodeficiency and evidence of opportunistic infections ( Pneumocystis , Candida , and Mycobacterium ). A single but unique founder virus, also present in the SHIV AD8-CE8J swarm stock, was transmitted to two animals following a single i.r. inoculation of approximately 3 50% animal infectious doses, which is close to the threshold required to establish infection in all exposed animals. Because the three new SHIV AD8 viruses are mucosally transmissible, exhibited tier 2 sensitivity to anti-HIV-1 neutralizing antibodies, deplete CD4 + T lymphocytes in vivo , and induce AIDS in macaques, they are eminently suitable as challenge viruses in vaccine experiments.

Evolution of the env gene in transmitted R5-tropic human immunodeficiency virus type 1 (HIV-1) strains is the most widely accepted mechanism driving coreceptor switching. In some infected individuals, however, a shift in coreceptor utilization can occur as a result of the reemergence of a cotransmitted, but rapidly controlled, X4 virus. The latter possibility was studied by dually infecting rhesus macaques with X4 and R5 chimeric simian simian/human immunodeficiency viruses (SHIVs) and monitoring the replication status of each virus using specific primer pairs. In one of the infected monkeys, both SHIVs were potently suppressed by week 12 postinoculation, but a burst of viremia at week 51 was accompanied by an unrelenting loss of total CD4 + T cells and the development of clinical disease. PCR analyses of plasma viral RNA indicated an env gene segment containing the V3 region from the inoculated X4 SHIV had been transferred into the genetic background of the input R5 SHIV by intergenomic recombination, creating an X4 virus with novel replicative, serological, and pathogenic properties. These results indicate that the effects of retrovirus recombination in vivo can be functionally profound and may even occur when one of the recombination participants is undetectable in the circulation as cell-free virus.

A new pathogenic R5-tropic simian/human immunodeficiency virus (SHIV) was generated following serial passaging in rhesus macaques. All 13 animals inoculated with SHIV AD8 passaged lineages experienced marked depletions of CD4 + T cells. Ten of these infected monkeys became normal progressors (NPs) and had gradual losses of both memory and naïve CD4 + T lymphocytes, generated antiviral CD4 + and CD8 + T cell responses, and sustained chronic immune activation while maintaining variable levels of plasma viremia (10 2 to 10 5 RNA copies/ml for up to 3 years postinfection [p.i.]). To date, five NPs developed AIDS associated with opportunistic infections caused by Pneumocystis carinii , Mycobacterium avium , and Campylobacter coli that required euthanasia between weeks 100 and 199 p.i. Three other NPs have experienced marked depletions of circulating CD4 + T lymphocytes (92 to 154 cells/μl) following 1 to 2 years of infection. When tested for coreceptor usage, the viruses isolated from four NPs at the time of their euthanasia remained R5 tropic. Three of the 13 SHIV AD8 -inoculated macaques experienced a rapid-progressor syndrome characterized by sustained plasma viremia of >1 × 10 7 RNA copies/ml and rapid irreversible loss of memory CD4 + T cells that required euthanasia between weeks 19 and 23 postinfection. The sustained viremia, associated depletion of CD4 + T lymphocytes, and induction of AIDS make the SHIV AD8 lineage of viruses a potentially valuable reagent for vaccine studies.

We investigated whether a 28-day course of potent antiretroviral therapy, initiated at a time point (48 h postinoculation) following simian immunodeficiency virus (SIV) inoculation when the acquisition of a viral infection was virtually assured, would sufficiently sensitize the immune system and result in controlled virus replication when treatment was stopped. The administration of tenofovir 48 h after SIV inoculation to six Mamu-A*01 -negative rhesus macaques did, in fact, potently suppress virus replication in all of the treated rhesus macaques, but plasma viral RNA rapidly became detectable in all six animals following its cessation. Unexpectedly, the viral set points in the treated monkeys became established at two distinct levels. Three controller macaques had chronic phase virus loads in the range of 1 × 10 3 RNA copies/ml, whereas three noncontroller animals had set points of 2 × 10 5 to 8 × 10 5 RNA copies/ml. All of the noncontroller monkeys died with symptoms of immunodeficiency by week 60 postinfection, whereas two of the three controller animals were alive at week 80. Interestingly, the three controller macaques each carried major histocompatibility complex class I alleles that previously were reported to confer protection against SIV, and two of these animals generated cytotoxic T-lymphocyte escape viral variants during the course of their infections.

Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines compared with animals that received nonrecombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. Continued analysis of these animals over the subsequent 9 years has shown that they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4 + T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env, and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4 + T cells. These data are consistent with a role for neutralizing antibodies in nonsterilizing protection from high viremia and associated memory CD4 + T-cell loss.

We and others have reported that the vast majority of virus-producing CD4 + T cells during the acute infection of rhesus macaques with simian immunodeficiency virus (SIV) or CXCR4 (X4)-using simian/human immunodeficiency viruses (SHIVs) exhibited a nonactivated phenotype. These findings have been extended to show that resting CD4 + T lymphocytes collected from SIV- or X4-SHIV-infected animals during the first 10 days of infection continue to release virus ex vivo. Furthermore, we observed high frequencies of integrated viral DNA (up to 5.1 × 10 4 DNA copies per 10 5 cells) in circulating resting CD4 + T cells during the first 10 days of the infection. Integration of SIV DNA was detected only in memory CD4 + T cells and SHIVs preferentially integrated into resting naïve CD4 + T cells. Taken together, these results show that during the acute infection large numbers of resting CD4 + T cells carry integrated nonhuman primate lentiviral DNA and are the major source of progeny virions irrespective of coreceptor usage. Prompt and sustained interventions are therefore required to block the rapid systemic dissemination of virus and prevent an otherwise fatal clinical outcome.

A human immunodeficiency virus type 1 (HIV-1) derivative (HIVNL-DT5R) containing sequences encoding a 7-amino-acid segment of CA and the entirevifgene from simian immunodeficiency virus (SIV) was previously shown to establish spreading infections in cultured macaque peripheral blood mononuclear cells. To assess its replicative and disease-inducing properties in vivo, HIVNL-DT5Rwas inoculated into pig-tailed macaques. HIVNL-DT5Rgenerated plasma viremia in all five of the monkeys and elicited humoral responses against all of the HIV-1 structural proteins but did not cause CD4+T-lymphocyte depletion or clinical disease. Additional adaptation will be required to optimize infectivity in vivo.

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif -deficient SIVagm virions. The presence of CypA in vif -defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif -deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5α independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif -sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.

Human immunodeficiency virus and simian immunodeficiency virus (SIV) induce a slow progressive disease, characterized by the massive loss of memory CD4 + T cells during the acute infection followed by a recovery phase in which virus replication is partially controlled. However, because the initial injury is so severe and virus production persists, the immune system eventually collapses and a symptomatic fatal disease invariably occurs. We have assessed CD4 + T-cell dynamics and disease progression in 12 SIV-infected rhesus monkeys for nearly 2 years. Three macaques exhibiting a rapid progressor phenotype experienced rapid and irreversible loss of memory, but not naïve, CD4 + T lymphocytes from peripheral blood and secondary lymphoid tissues and died within the first 6 months of virus inoculation. In contrast, SIV-infected conventional progressor animals sustained marked but incomplete depletions of memory CD4 + T cells and continuous activation/proliferation of this T-lymphocyte subset. This was associated with a profound loss of naïve CD4 + T cells from peripheral blood and secondary lymphoid tissues, which declined at rates that correlated with disease progression. These data suggest that the persistent loss of memory CD4 + T cells, which are being eliminated by direct virus killing and activation-induced cell death, requires the continuous differentiation of naïve into memory CD4 + T cells. This unrelenting replenishment process eventually leads to the exhaustion of the naïve CD4 + T-cell pool and the development of disease.

Current antiretroviral therapy (ART) is not sufficient to completely suppress disease progression in the CNS, as indicated by the rising incidence of HIV-1-associated neurocognitive disorders (HAND) among infected individuals on ART. It is not clear why some HIV-1-infected patients develop HAND, despite effective repression of viral replication in the circulation. SIV-infected nonhuman primate models are widely used to dissect the mechanisms of viral pathogenesis in the CNS. Here, we identified 4 amino acid substitutions in the cytoplasmic tail of viral envelope glycoprotein gp41 of the neurovirulent virus SIVsm804E that enhance replication in macrophages and associate with enhanced antagonism of the host restriction factor BM stromal cell antigen 2 (BST-2). Rhesus macaques were inoculated with a variant of the parental virus SIVsmE543-3 that had been engineered to contain the 4 amino acid substitutions present in gp41 of SIVsm804E. Compared with WT virus-infected controls, animals infected with mutant virus exhibited higher viral load in cerebrospinal fluid. Together, these results are consistent with a potential role for BST-2 in the CNS microenvironment and suggest that BST-2 antagonists may serve as a possible target for countermeasures against HAND.

The simian immunodeficiency viruses (SIV) naturally infect a wide range of African primates, including African green monkeys (AGM). Despite moderate to high levels of plasma viremia in naturally infected AGM, infection is not associated with immunodeficiency. We recently reported that SIVagmVer90 isolated from a naturally infected vervet AGM induced AIDS following experimental inoculation of pigtailed macaques. The goal of the present study was to evaluate the replication of this isolate in two species of AGM, sabaeus monkeys ( Chlorocebus sabaeus ) and vervets ( C. pygerythrus ). Inoculation of sabaeus AGM with SIVagmVer90 resulted in low and variable primary and set-point viremia (2 to 10 4 copies/ml). In contrast, inoculation of vervet AGM with either SIVagmVer90 or blood from a naturally infected vervet (Ver1) resulted in high primary viremia and moderate plateau levels, similar to the range seen in naturally infected vervets from this cohort. CD4 + T cells remained stable throughout infection, even in AGM with persistent high viremia. Despite the lack of measurable lymphadenopathy, infection was associated with an increased number of Ki-67 + T cells in lymph node biopsies, consistent with an early antiviral immune response. The preferential replication of SIVagmVer in vervet versus sabaeus AGM shows that it is critical to match AGM species and SIV strains for experimental models of natural SIV infection.

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of env in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.

For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4+ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency., Author Summary Primate lentiviruses, such as HIV and its SIV simian relative, encode accessory proteins that suppress cellular restriction factors interfering with efficient replication. One of these, designated Vpx, is produced in infected cells by HIV-2 and some SIV strains, which cause endemic infections in African monkeys. The primary function of Vpx has long been thought to facilitate infectivity in dendritic cells and macrophage by degrading the Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1), which restricts virus replication in these cells. Using SIVmac carrying a mutated Vpx gene with a single amino acid change that prevents it from binding to DCAF1 and subsequently mediating the degradation of SAMHD1, we show that virus infection of CD4+ T lymphocytes is markedly compromised both in vitro and in vivo. The SIV Vpx mutant is severely attenuated in establishing new infections in inoculated rhesus monkeys, in producing high levels of virus progeny, in degrading SAMHD1 in memory CD4+ T cell in infected animals, and in inducing symptomatic disease. Thus, although once considered to be only critical for optimal replication in macrophage based on earlier studies performed with cultured cells, the SIV Vpx protein is functionally important in vivo for establishing the primary infection in rhesus macaques, sustaining high levels of virus replication in CD4+ T lymphocytes, and promoting the onset of symptomatic immunodeficiency.

Tripartite motif-containing protein 5α (TRIM5α) is considered to be a potential target for cell-based gene modification therapy against human immunodeficiency virus type 1 (HIV-1) infection. In the present study, we used a relevant rhesus macaque model of infection with simian immunodeficiency virus from sooty mangabey (SIVsm) to evaluate the effect of TRIM5α restriction on clinical outcome. For macaques expressing a restrictive TRIM5 genotype, the disease outcomes of those infected with the wild-type TRIM-sensitive SIVsm strain and those infected with a virus with escape mutations in the capsid were compared. We found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, supporting the feasibility of exploiting TRIM5α as a target for gene therapy against HIV-1. Furthermore, we also found that preservation of memory CD4 T cells was associated with protection by TRIM5α restriction, suggesting memory CD4 T cells or their progenitor cells as an ideal target for gene modification. Despite the significant effect of TRIM5α restriction on survival, SIV escape from TRIM5α restriction was also observed; therefore, this may not be an effective stand-alone strategy and may require combination with other targets. IMPORTANCE Recent studies suggest that it may be feasible not only to suppress viral replication with antiviral drugs but also potentially to eliminate or “cure” human immunodeficiency virus (HIV) infection. One approach being explored is the use of gene therapy to introduce genes that can restrict HIV replication, including a restrictive version of the host factor TRIM5α. TRIM5 was identified as a factor that restricts HIV replication in macaque cells. The rhesus gene is polymorphic, and some alleles are restrictive for primary SIVsm isolates, although escape mutations arise late in infection. Introduction of these escape mutations into the parental virus conferred resistance to TRIM5 on macaques. The present study evaluated these animals for long-term outcomes and found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, suggesting that this could be a valid gene therapy approach that could be adapted for HIV.

We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIVDH12R-Clone 7, like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIVDH12R-Clone 8experience only partial and transient losses of CD4+T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+and CD20+lymphocytes in suppressing the replication of the attenuated SHIVDH12R-Clone 8and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+or CD20+cells during the SHIVDH12R-Clone 8acute infection resulted in the rapid, complete, and irreversible loss of CD4+T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+cells; SHIVDH12R-Clone 8was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+and CD20+B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.

Simian immunodeficiency virus (SIV) is known to result in an asymptomatic infection of its natural African monkey host. However, some SIV strains are capable of inducing AIDS-like symptoms and death upon experimental infection of Asian macaques. To further investigate the virulence of natural SIV isolates from African monkeys, pig-tailed (PT) macaques were inoculated intravenously with either of two recently discovered novel lentiviruses, SIVlhoest and SIVsun. Both viruses were apparently apathogenic in their natural hosts but caused immunodeficiency in PT macaques. Infection was characterized by a progressive loss of CD4 + lymphocytes in the peripheral blood and lymph nodes, generalized lymphoid depletion, a wasting syndrome, and opportunistic infections, such as Mycobacterium avium or Pneumocystis carinii infections. However, unlike SIVsm/mac infection of macaques, SIVlhoest and SIVsun infections in PT macaques were not accompanied by high viral loads during the chronic disease stage. In addition, no significant correlation between the viral load at set point (12 weeks postinfection) and survival could be found. Five out of eight SIVlhoest-infected and three out of four SIVsun-infected macaques succumbed to AIDS during the first 5 years of infection. Thus, the survival of SIVsun- and SIVlhoest-infected animals was significantly longer than that of SIVagm- or SIVsm-infected macaques. All PT macaques maintained strong SIV antibody responses despite progression to SIV-induced AIDS. The development of immunodeficiency in the face of low viremia suggests that SIVlhoest and SIVsun infections of macaques may model unique aspects of the pathogenesis of human immunodeficiency virus infection in humans.

In vivo, the principal target of human immunodeficiency virus type 1 (HIV-1) is the CD4+ T cell. Over time, virus-induced elimination and/or dysfunction of this T-lymphocyte subset, whether caused directly or indirectly, leads to clinical disease in infected individuals (10). Like all other lentiviruses, HIV-1 is also able to infect macrophage, a property recognized since the beginning of the AIDS epidemic (21, 50). The pathogenic consequences of HIV-1-infected macrophages is best exemplified by the AIDS dementia complex in which viral RNA expressed in microglia within the central nervous system may cause severe neuronal injury leading to encephalopathy (24). In addition, because of their reported resistance to the cytopathic effects elicited by HIV-1 and long life span, tissue macrophages have also been considered to be an important reservoir of virus (36). In this regard, macrophages have been reported to be a prominent source of virus during the late stages of disease, especially in conjunction with opportunistic infections (35). HIV-1 infection of macrophages in vivo has been logistically difficult to study. For example, it is not currently known whether monocyte precursors are initially infected in the bone marrow and only begin to produce virus after their migration to and differentiation in specific tissues or whether they become infected after they reach their final tissue of residence. Current understanding about the dynamics of virus production by tissue macrophage is similarly limited: are progeny virions generated as a result of de novo infections or by the continuous release of particles by long-lived cells? Consequently, most present knowledge about HIV-1 infection of macrophage accrues from an in vitro surrogate: monocyte-derived macrophage (MDM). MDM have been useful for identifying so-called macrophage-tropic (M-tropic) HIV-1 strains. M-tropic strains infect MDM and primary CD4+ T lymphocytes but not most human T-cell lines in vitro, fail to induce syncytium formation, and can be recovered from infected persons during all phases of their disease (7, 13, 42). Prototypic M-tropic strains of HIV-1 use the CCR5 chemokine receptor for cell entry (1, 11). From studies of highly pathogenic SHIVs that irreversibly and systemically deplete CD4+ T lymphocytes in rhesus monkeys within weeks of virus inoculation, we previously reported that tissue macrophages are able to sustain high levels of plasma viremia (>106 RNA copies/ml) in the virtual absence of CD4+ T cells (16). It was subsequently found that viral variants, circulating in the plasma during the macrophage phase of SHIV infections, carried gp120 V2 changes (specific double amino acid deletions and the loss of a conserved glycosylation site) (19). Some of these SHIV variants had also acquired the capacity to infect alveolar macrophages (AM). In the present study, the gp120 structure, cell tropism, and coreceptor utilization properties of macrophage-phase simian immunodeficiency virus/HIV chimeras (SHIVs), isolated directly from lymphoid tissues, were examined. In contrast to plasma virus, the tissue-associated SHIVs bore envelope glycoproteins that were genetically more heterogeneous and contained specific changes primarily within V1 and V2. Small molecule coreceptor-targeted inhibitors, specific for CCR5 or CXCR4, were used to assess the chemokine receptor usage by (i) the starting highly pathogenic, T-cell-deleting SHIVs and the (ii) late-phase M-tropic SHIV variants. Blockade of CXCR4 potently suppressed infection of rhesus monkey PBMC by both viruses, whereas CCR5 targeted inhibitors had little if any effect. Infection of macaque AM by M-tropic SHIVs was also suppressed by CXCR4 not by CCR5 specific inhibitors. Thus, the acquisition of macrophage tropism by SHIVs, present in late-phase monkeys, is not accompanied by a switch in chemokine coreceptor usage.

Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+T-lymphocyte loss.

Five potent and broadly anti-HIV neutralizing monoclonal antibodies are able to block infection by two different SHIVs in monkeys. The authors show that antibodies targeting the outer glycan coat were the most effective and determined that titers of roughly 1:100 protected half the animals., It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti–HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (∼1:100) and potentially achievable by vaccination.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239gag-poland HIV-1 89.6envelicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV DH12 ) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups ( P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV DH12 strongly correlated with the level of NAbs directed against the virus on the day of challenge ( P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV DH12 challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) ( P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

The highly pathogenic simian immunodeficiency virus/HIV type 1 (SHIV) chimeric virus SHIV DH12R induces a systemic depletion of CD4 + T lymphocytes in rhesus monkeys during the initial 3–4 weeks of infection. Nonetheless, high levels of viral RNA production continue unabated for an additional 2–5 months. In situ hybridization and immunohistochemical analyses revealed that tissue macrophage in the lymph nodes, spleen, gastrointestinal tract, liver, and kidney sustain high plasma virus loads in the absence of CD4 + T cells. Quantitative confocal immunofluorescence analysis indicated that greater than 95% of the virus-producing cells in these tissues are macrophage and less than 2% are T lymphocytes. Interestingly, the administration of a potent reverse transcriptase inhibitor blocked virus production during the early T cell phase but not during the later macrophage phase of the SHIV DH12R infection. When interpreted in the context of HIV-1 infections, these results implicate tissue macrophage as an important reservoir of virus in vivo . They become infected during the acute infection, gradually increase in number over time, and can be a major contributor to total body virus burden during the symptomatic phase of the human infection.

Virus–specific antibodies protect individuals against a wide variety of viral infections1–7. To assess whether human immunodeficiency virus type 1 (HIV–1) envelope–specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV–1 isolates, and used this for passive immunization of pig–tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian–human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV–1DH12, a dual–tropic primary virus isolate. Here we show that anti–SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody–mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV–challenged macaques was in the range of 1:5–1:8. The HIV–1–specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non–neutralizing anti–HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

Immunologic reagents and methodology are essential to develop further the woodchuck and woodchuck hepatitis virus (WHV) as a model of immune response, inflammation, and immunotherapy in hepatitis B virus (HBV) infection. Partial cDNA clones for the woodchuck CD3ϵ marker of T cells (536 bp) and for selected woodchuck cytokines were developed, including IL-1β (332 bp), IL-2 (249 bp), IL-4 (205 bp), IL-10 (476 bp), IFN-γ (476 bp), and TNF-α (381 bp). This panel of markers includes sets to measure RNAs for T cells (CD3ϵ), immune response induction (IL-1β, IL-2), TH subsets (TH1, IL-2/IFN-γ vs. TH2, IL-4/IL-10), and effector molecules that regulate hepadnavirus replication and liver injury (IFN-γ, TNF-α). Primers representing highly conserved segments of genes from other species were used to derive the partial cDNA clones. Target RNA was obtained from woodchuck peripheral blood mononuclear cells (PBMC) that were stimulated in vitro with ConA, LPS, and human rIL-2. The cDNA clones were validated by 1) comparison with other species for homologies in the nucleotide and predicted amino acid sequences and 2) a first generation assay demonstrating induction of the respective RT-PCR products in stimulated woodchuck PBMC. The corresponding RNAs were also detectable in most cases in the total RNA from the livers of uninfected and WHV-infected woodchucks and differential expression of IFN-γ and TNF-α RNAs was suggested. Second generation, semi-quantitative assays for the RNAs were validated using RT-PCR and dot-blot hybridization with 32P-oligomers derived from the internal sequences of the respective clones. Continued study of the woodchuck immune response to WHV infection using these assays will provide insight into the kinetics and immune mechanisms that initiate and maintain chronic hepadnavirus infection and, hence, enable development of improved immunotherapies for established chronic HBV infection. J. Med. Virol. 53:85–95, 1997. © 1997 Wiley-Liss, Inc.

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9-14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7 days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5 weeks in some long-term chronically SHIV-infected animals with low CD4(+) T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5TFP/TFP or TRIM5Cyp/TFP. Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5Cyp. Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIMTFP alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates., Author Summary Human immunodeficiency virus (HIV) resulted from the transmission of simian immunodeficiency viruses (SIV) from nonhuman primates followed by adaptation and expansion as a pandemic in humans. This required the virus to overcome a variety of intrinsic host restriction factors in humans in order to replicate efficiently. Similarly, SIV encounters restriction factors upon cross-species transmission between nonhuman primates, specifically from a natural host species such as sooty mangabey monkeys to rhesus macaques. Previously we observed significant differences in the levels of virus replication of SIV among rhesus macaques due to subtle differences in one of these restriction factors, TRIM5 among individual macaques. Although a restrictive version of TRIM5 resulted in lower viremia, we also observed that the virus spontaneously mutated in the viral capsid gene and that these mutations were associated with escape from TRIM5 restriction. In the present study, we found that introduction of these escape mutations into the parental virus confers resistance to TRIM5 both in tissue culture and in macaques. These studies provide direct evidence that TRIM5 is a critical factor influencing the cross-species transmission of SIV in primates.

Fresh human immunodeficiency virus type 1 (HIV-1) isolates from patients with AIDS were screened for infectivity in chimpanzee peripheral blood mononuclear cells (PBMC) to identify strains potentially able to generate high virus loads in an inoculated animal. Only 3 of 23 isolates obtained were infectious in chimpanzee cells. Of these three, only one (HIV-1DH12) was able to initiate a productive infection in PBMC samples from all 25 chimpanzees tested. HIV-1DH12 tissue culture infections were characterized by extremely rapid replication kinetics, profound cytopathicity, and tropism for chimp and human PBMC, primary human macrophage, and several human T-cell lines. An infection was established within 1 week of inoculating a chimpanzee with 50 50% tissue culture infective doses of HIV-1DH12; cell-free virus was recovered from the plasma at weeks 1, 2, and 4 and was associated with the development of lymphadenopathy. Virus loads during the primary infection and at 6 months postinoculation were comparable to those reported in HIV-1-seropositive individuals.

The matrix protein of human immunodeficiency virus type 1 is encoded by the amino-terminal portion of the Gag precursor and is postulated to be involved in a variety of functions in the virus life cycle. To define domains and specific amino acid residues of the matrix protein that are involved in virus particle assembly, we introduced 35 amino acid substitution mutations in the human immunodeficiency virus type 1 matrix protein. Using reverse transcriptase and radioimmunoprecipitation analyses and transmission electron microscopy, we assessed the mutants for their ability to form virus particles and to function in the infection process. This study has identified several domains of the matrix protein in which single amino acid substitutions dramatically reduce the efficiency of virus particle production. These domains include the six amino-terminal residues of matrix, the region of matrix between amino acids 55 and 59, and the region between amino acids 84 and 95. Single amino acid substitutions in one of these domains (between matrix amino acids 84 and 88) result in a redirection of the majority of virus particle formation to sites within cytoplasmic vacuoles.

Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains three binding sites for the transcriptional factor Sp1. In order to investigate the role that the Sp1-binding sites play in regulation of HIV replication, we have introduced a deletion of all three Sp1-binding sites into the LTR of an infectious molecular clone of HIV. Viral stocks have been prepared from this mutant virus, designated dl-Sp, and these stocks have been used to study its replicative ability in human T cells. The dl-Sp virus replicated efficiently in MT4 cells and in phytohemagglutinin-stimulated human peripheral blood lymphocytes, but it replicated poorly and with delayed kinetics in A3.01 (CEM) T cells unless those cells had been treated with the cytokine tumor necrosis factor alpha. Gel retardation assays to study the levels of DNA-binding proteins present in these cells showed that NF-kappa B activity could be detected in the nuclei of MT4 cells but not in A3.01 cells unless they had been treated with tumor necrosis factor alpha. Thus, the presence of NF-kappa B activity appeared to be required for efficient replication of an HIV whose LTR Sp1-binding sites had been deleted. This suggests that NF-kappa B can functionally compensate for Sp1 in activating HIV replication. The HIV LTR is therefore similar to the promoter-enhancer units of other viruses in that it is composed of multiple functional elements that may contribute differently to viral replication depending on the levels of DNA-binding proteins present in the target cells.

Unlike prototypical lentiviruses like visna and caprine arthritis-encephalitis viruses, which are mainly macrophage tropic (M-tropic), primate lentiviruses primarily target CD4 + T lymphocytes. We previously reported that during the late phase of highly pathogenic chimeric simian/human immunodeficiency virus (SHIV) infections of rhesus macaques, when CD4 + T cells have been systemically eliminated, high levels of viremia are maintained from productively infected macrophages. The availability of several different M-tropic SHIVs from such late-stage immunocompromised animals provided the opportunity to assess whether they might contribute to the immune deficiency induced by their T-cell-tropic parental viruses or possibly cause a distinct disease based on their capacity to infect macrophages. Pairs of rhesus monkeys were therefore inoculated intravenously with six different M-tropic SHIV preparations, and their plasma viral RNA loads, circulating lymphocyte subset numbers, and eventual disease outcomes were monitored. Only one of these six M-tropic SHIVs induced any disease; the disease phenotype observed was the typical rapid, complete, and irreversible depletion of CD4 + T cells induced by pathogenic SHIVs. An analysis of two asymptomatic monkeys, previously inoculated with an M-tropic SHIV recovered directly from alveolar macrophages, revealed that this inoculum targeted alveolar macrophages in vivo, compared to a T-cell-tropic virus, yet no clinical disease occurred. Although one isolate did, in fact, induce the prototypical rapid, irreversible, and complete loss of CD4 + T cells, indicating that M-tropism and pathogenicity may not be inversely related, the majority of M-tropic SHIVs induced no clinical disease in immunocompetent macaques.

Rapid progression to disease is observed in patients infected with human immunodeficiency virus (HIV) (9, 13, 30, 38, 40, 60) and macaques experimentally infected with simian immunodeficiency virus (SIV) (15, 18, 21, 22, 24, 31, 44, 47, 52, 64). In both cases, the level of plasma viremia is significantly higher than in conventional progressors, and the cellular and humoral immune responses are very weak and only transiently observed. In this study, as well as prior studies, we defined rapid progressors (RPs) as SIV-inoculated macaques with persistent antigenemia that failed to develop or maintain antibody responses and showed clinical deterioration that necessitated euthanasia before 6 months of inoculation. Recent studies have shown that SIV-infected RP macaques suffer an early massive loss of memory CD4+ T cells, presumably due to virus-induced destruction (24, 31, 34, 37, 44, 45, 52, 61). The loss of memory CD4+ T cells is profound and irreversible in RP macaques, suggesting a strong correlation between the maintenance of CD4+ memory T cells and rapid progression to disease (31, 44, 45, 52). Although these studies of RPs using the SIV model are useful for understanding the mechanism of rapid progression in HIV infection, it is not clear why the depletion of memory CD4+ T cells is so profound in RP macaques and how they develop disease rapidly after the depletion. Recent studies have shown that RP macaques can be distinguished from conventional progressors in terms of pathological manifestations (2, 18, 21, 31). RP macaques uniformly exhibit SIV encephalitis and pneumonia, the presence of multinucleated giant cells, and a predominance of SIV-expressing macrophages in nonlymphoid tissues, such as lung and brain. In contrast, conventional progressor macaques show the pathological features more analogous to AIDS in humans, i.e., the predominance of opportunistic infections. However, the definitive hallmark of rapid progression is the failure to maintain SIV-specific immune responses (18, 21, 64). RP macaques mount an initial humoral and cellular immune response at the appropriate time following infection, but these responses wane rapidly within the first 3 to 4 weeks of infection (21). This immune defect is observed not only in immunity to SIV, but also in immune responses against unrelated antigens, including recall antigens (tetanus toxoid) or new antigen (hepatitis A virus). This profound and global immune failure is consistent with the early loss in T-helper function caused by a massive loss of memory CD4+ T cells (24, 31, 34, 37, 44, 45, 52, 61) and may be critical for the subsequent rapid progression of disease. Despite the lack of immune pressure, the envelope of SIV undergoes unique molecular evolution in RP macaques (8, 31). Sequence analysis of viruses in three SIVsmE543-3-infected RP macaques (8) revealed a unique convergent pattern of substitutions in env, including the loss of a highly conserved potential N-linked glycosylation (PNG) site in the V1-V2 region (N158D/S or S160N/G), substitutions in the V3 analog (P337T/S/H/L and R348W), and substitutions in the highly conserved GDPE motif (G386R and D388N/V). These RP-specific mutations are associated with the acquisition of CD4-independent usage of CCR5 in cell fusion assays (8, 11, 57). In addition, mutational analyses have shown that, like in HIV type 1 (HIV-1) (33, 46), the GDPE motif and/or V3 loop analog of SIV are important for cell tropism and interactions with CD4 and coreceptors (11, 20, 49, 57). These data suggested that RP-specific mutations were selected in RP macaques to adapt to a specific microenvironment in RP macaques through altered cell tropism and receptor usage. In order to study the role of these variants with RP-specific mutations in rapid progression, seven infectious molecular clones were generated from the terminal-phase plasma of an RP macaque (31). We initially predicted that these clones would show an advantage over parental SIV in terms of replication efficacy or cell tropism that would explain the high viral loads in the plasma and tissues of RP macaques. However, clones with RP-specific mutations replicated less efficiently than their parent, SIVsmE543-3, in primary rhesus peripheral blood mononuclear cells and macrophages (31). From these in vitro analyses, it was not clear why RP variants predominated in RP macaques or whether RP variants contributed to the development of rapid disease progression. The goal of the present study was to analyze the biological properties of RP variants in vivo using infectious molecular clones. For this purpose, we chose SIVsmH635FC, since it was closest in terms of sequence to the consensus sequence of plasma virus in the donor animal. Macaques were inoculated with SIVsmH635FC alone or in combination with parental SIVsmE543-3, and virus replication, changes in CD4+ T-cell subsets in blood and mucosal sites, antibody responses, and the stability of RP-specific mutations were evaluated. The coinfected animals allowed us to evaluate the in vivo fitness of SIVsmH635FC relative to that of SIVsmE543-3.

Unlike HIV-1 and simian immunodeficiency virus (SIV), which induce a slow, unrelenting loss of immune function spanning several years, highly pathogenic simian–human immunodeficiency viruses (SHIVs) induce a rapid, complete, and irreversible depletion of CD4 + T lymphocytes in rhesus monkeys within weeks of infection, leading to death from immunodeficiency. We recently reported that, because these SHIVs exclusively use the CXCR4 coreceptor for cell entry, they target naïve CD4 + T cells for depletion in infected monkeys, whereas SIVs, which use CCR5, not CXCR4, cause the selective loss of memory CD4 + T lymphocytes in vivo . Here we show both by DNA PCR analyses and infectivity assays, using live sorted CD4 + T lymphocyte subsets, that 30–90% of circulating naïve cells were productively infected by day 10 after inoculation. This result implies that direct cell killing, not bystander apoptosis, is responsible for the massive loss of CD4 + T cells in the X4-tropic SHIV model. Furthermore, we directly demonstrate that >96% of virus producing cells did not express the Ki-67 proliferation marker on day 10 after inoculation using confocal microscopic analysis of lymph nodes samples. This finding is consistent with the prodigious levels of plasma viremia measured during acute X4-tropic SHIV infections of macaques being generated almost entirely by resting naïve CD4 + T cells.

Simian immunodeficiency virus from African green monkeys (SIVagm) results in asymptomatic infection in its natural host species. The virus is not inherently apathogenic, since infection of pigtailed (PT) macaques ( Macaca nemestrina ) with one isolate of SIVagm results in an immunodeficiency syndrome characterized by progressive CD4 + -T-cell depletion and opportunistic infections. This virus was passaged once in a PT macaque and, thus, may not be entirely reflective of the virulence of the parental strain. The goal of the present study was to assess the pathogenicity of the PT-passaged isolate (SIVagm9063) and two primary SIVagm isolates in PT macaques, including the parental strain of the PT-passaged variant. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4 + -T-cell subsets and remain healthy. The plateau levels among SIVagm90- and SIVagm9063-inoculated macaques varied widely from 100 to 1 million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4 + -T-cell loss at 6 months postinfection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques ( P values of + -T-cell loss by 6 months postinfection correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. The inherent pathogenicity of natural SIVagm isolates varies, but such natural isolates are capable of inducing AIDS in macaques without prior macaque passage.

The virus infectivity factor (Vif) is a protein encoded by most primate lentiviruses. Recent evidence suggests that HIV-1 Vif reduces the intracellular levels of the host cytidine deaminase APOBEC3G (Apo3G) and inhibits its packaging into virions. These functions of Vif are thought to be species-specific. Accordingly, HIV-1 Vif can target only human Apo3G (hApo3G), whereas, African green monkey simian immunodeficiency virus (SIVagm) Vif can inhibit African green monkey but not human Apo3G. Consistent with this, we found that SIVagm Vif does not affect the stability of exogenously and endogenously expressed hApo3G and does not prevent packaging of exogenous and endogenous hApo3G into SIVagm virions. Nevertheless, SIVagm Vif supported spreading infection of SIVagm virus in the hApo3G-positive human A3.01 T cell line and rescued infectivity of viruses produced from Apo3G-expressing HeLa cells. Sequence analysis verified that SIVagm Vif inhibited the accumulation of hApo3G-induced mutations, suggesting that SIVagm Vif is indeed active in human cells. Our data suggest that SIVagm Vif can inhibit hApo3G activity without inducing its intracellular degradation or preventing its packaging into virions.

In contrast to simian immunodeficiency viruses (SIVs), which induce immunodeficiency over a 1- to 3-year period, highly pathogenic simian-human immunodeficiency viruses (SHIVs) cause a complete, irreversible, and systemic depletion of CD4+T lymphocytes in rhesus monkeys within weeks of infection. By using small-molecule competitors specific for CCR5 and CXCR4 inex vivoassays, we found that highly pathogenic SHIVDH12Rexclusively uses CXCR4 for infection of rhesus peripheral blood mononuclear cells, whereas SIVmac239and SIVsmE543use CCR5 for entry into the same cells. During the period of peak virus production in SHIVDH12R- or SHIV89.6P-infected rhesus monkeys, massive elimination of CXCR4+naïve CD4+T cells occurred. In contrast, circulating CCR5+memory CD4+T cells were selectively depleted in rapidly progressing SIV-infected monkeys. At the time of their death, two SIV rapid progressors had experienced a nearly complete loss of the memory CD4+T cell subset from the blood and mesenteric lymph nodes. Thus, pathogenic SHIVs and SIVs target different subsets of CD4+T cellsin vivo, with the pattern of CD4+T lymphocyte depletion being inextricably linked to chemokine receptor use. In the context of developing an effective prophylactic vaccine, which must potently control virus replication during the primary infection, regimens that suppress SHIVs might not protect monkeys against SIV or humans against HIV-1.

One of three full-length infectious molecular clones of SHIV DH12R , designated SHIV DH12R-CL-7 and obtained from productively infected rhesus monkey peripheral blood mononuclear cells, directed rapid and irreversible loss of CD4 + T cells within 3 weeks of its inoculation into Indian rhesus monkeys. Induction of complete CD4 + T-cell depletion by SHIV DH12R-CL-7 was found to be dependent on inoculum size. The acquisition of this pathogenic phenotype was accompanied by the introduction of 42 amino acid substitutions into multiple genes of parental nonpathogenic SHIV DH12 . Transfer of the entire SHIV DH12R-CL-7 env gene into the genetic background of nonpathogenic SHIV DH12 failed to confer the rapid CD4 + T-lymphocyte-depleting syndrome; similarly, the substitution of gag plus pol sequences from SIV smE543 for analogous SIV mac239 genes in SHIV DH12R-CL-7 attenuated the pathogenic phenotype. Amino acid changes affecting multiple viral genes are necessary, but insufficient by themselves, to confer the prototypically rapid and irreversible CD4 + T-cell-depleting phenotype exhibited by molecularly cloned SHIV DH12R-CL-7 .