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Simian-human immunodeficiency virus (SHIV) infection provides a relevant animal model to study HIV-1 neutralization breadth. With previously identified SHIVSF162P3N infected rhesus macaques that did or did not develop neutralization breadth, we characterized the transmitted/founder viruses and initial autologous/homologous neutralizing antibodies in these animals. The plasma viral load and blood CD4 count did not distinguish macaques with and without breadth, and only one tested homologous envelope clone revealed a trend for macaques with breadth to favor an early homologous response. In two macaques with breadth, GB40 and FF69, infected with uncloned SHIVSF162P3N, multiple viral variants were transmitted, and the transmitted variants were not equal in neutralization sensitivity. The targets of initial autologous neutralizing antibodies, arising between 10 and 20 weeks post infection, were mapped to N462 glycan and G460a in gp120 V5 in GB40 and FF69, respectively. Although it is unclear whether these targets are related to later neutralization breadth development, the G460a target but not N462 glycan appeared more common in macaques with breadth than those without. Longitudinal plasmas revealed 2–3 sequential waves of neutralizing antibodies in macaques with breadth, implicating that 3 sequential envelope variants, if not more, may be required for the broadening of HIV-1 neutralizing antibodies.

Macrophage (mac)-tropic human immnunodeficiency virus type 1 (HIV-1) and simian immnunodeficiency virus (SIV) in brain are associated with neurological disease. Mac-tropic HIV-1 evolves enhanced CD4 interactions that enable macrophage infection via CD4, which is in low abundance. In contrast, mac-tropic SIV is associated with CD4-independent infection via direct CCR5 binding. Recently, mac-tropic simian-human immunodeficiency virus (SHIV) from macaque brain was also reported to infect cells via CCR5 without CD4. Since SHIV envelope proteins (Envs) are derived from HIV-1, we tested more than 100 HIV-1 clade B Envs for infection of CD4-negative, CCR5+ Cf2Th/CCR5 cells. However, no infection was detected. Our data suggest that there are differences in the evolution of mac-tropism in SIV and SHIV compared to HIV-1 clade B due to enhanced interactions with CCR5 and CD4, respectively.

Objective Gender differences in immune response and the rate of disease progression in HIV-infected individuals have been reported but the underlying mechanism remains unclear, in part because of the lack of relevant animal models. Here, we report a novel nonhuman primate model for investigation of sex disparity in HIV disease progression. Design/methods Viral load and rate of disease progression were evaluated in rhesus macaques infected intrarectally with lineage-related subtype C R5 simian HIVs. Cytokine/chemokine levels in rectal swab eluates, and bacterial species adherent to the swabs and in the feces were determined. Results Simian HIV-infected female rhesus macaques progressed faster to AIDS than male macaques, recapitulating the sex bias in HIV-1 disease in humans. There were no significant differences in the levels of soluble immune mediators in the rectal mucosa of naive female and male macaques. However, an exploratory longitudinal study in six infected macaques indicates that the female macaques mounted an earlier and more robust proinflammatory skewed rectal immune response to infection. Moreover, expansion of Proteobacteria that increase in other intestinal inflammatory disorders was significantly higher in the rectal mucosa of female than male macaques during acute infection. Conclusion These findings suggest that sex differences in local innate immune activation and compositional shifts in the gut microbiota could be the drivers of increased disease susceptibility in female macaques. Further studies with this novel nonhuman primate model of HIV infection could lead to innovative research on gender differences, which may have important therapeutic implications for controlling disease in infected men as well as women.

BACKGROUND HIV-1 is known to adapt to the local environment in its usage of receptors, and it can become CD4 independent in the brain where the receptor is scarce. This adaptation is through amino acid variations, but the patterns of such variation are not yet well understood. Given that infection of long-lived CD4-low and CD4-negative cells in anatomical compartments such as the brain expands cell tropism in vivo and may serve as potential viral reservoirs that pose challenge for HIV eradication, understanding the evolution to CD4 independence and envelope conformation associated with infection in the absence of CD4 will not only broaden our insights into HIV pathogenesis but may guide functional cure strategies as well. METHODS We characterize, by site-directed mutagenesis, neutralization assay, and structural analysis, a pair of CD4-dependent (cl2) and CD4-independent (cl20) envelopes concurrently isolated from the cerebral spinal fluid of an SHIV-infected macaque with neurological AIDS and with minimum sequence differences. RESULTS Residues different between cl2 and cl20 are mapped to the V1V2 and surrounding regions. Mutations of these residues in cl2 increased its CD4 independence in infection, and the effects are cumulative and likely structural. CONCLUSIONS Our data suggested that the determinants of CD4 independence in vivo mapped principally to V1V2 of gp120 that can destabilize the apex of the envelope spike, with an additional change in V4 that abrogated a potential N-linked glycan to facilitate movement of the V1V2 domain and further expose the coreceptor-binding site.

Human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) is characterized by replication in macrophages or brain microglia that express low levels of the CD4 receptor and is the cause of HIV-associated dementia and related cognitive and motor disorders that affect 20 to 30% of treatment-naive patients with AIDS. Independent viral envelope evolution in the brain has been reported, with the need for robust replication in resident CD4 low cells, as well as CD4-negative cells, such as astrocytes, proposed as a major selective pressure. We previously reported giant-cell encephalitis in subtype B and C R5 simian-human immunodeficiency virus (SHIV)-infected macaques (SHIV-induced encephalitis [SHIVE]) that experienced very high chronic viral loads and progressed rapidly to AIDS, with varying degrees of macrophage or microglia infection and activation of these immune cells, as well as astrocytes, in the CNS. In this study, we characterized envelopes (Env) amplified from the brains of subtype B and C R5 SHIVE macaques. We obtained data in support of an association between severe neuropathological changes, robust macrophage and microglia infection, and evolution to CD4 independence. Moreover, the degree of Env CD4 independence appeared to correlate with the extent of astrocyte infection in vivo . These findings further our knowledge of the CNS viral population phenotypes that are associated with the severity of HIV/SHIV-induced neurological injury and improve our understanding of the mechanism of HIV-1 cellular tropism and persistence in the brain. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes in the brain has been suggested to be important in HIV persistence and neuropathogenesis but has not been definitively demonstrated in an animal model of HIV-induced encephalitis (HIVE). Here, we describe a new nonhuman primate (NHP) model of R5 simian-human immunodeficiency virus (SHIV)-induced encephalitis (SHIVE) with several classical HIVE features that include astrocyte infection. We further show an association between severe neuropathological changes, robust resident microglia infection, and evolution to CD4 independence of viruses in the central nervous system (CNS), with expansion to infection of truly CD4-negative cells in vivo . These findings support the use of the R5 SHIVE models to study the contribution of the HIV envelope and viral clades to neurovirulence and residual virus replication in the CNS, providing information that should guide efforts to eradicate HIV from the body.

Most human immunodeficiency virus (HIV) transmissions are initiated with CCR5 (R5)-using viruses across mucosal surfaces, with the majority in regions where HIV type 1 (HIV-1) clade C predominates. Mucosally transmissible, highly replication competent, pathogenic R5 simian-human immunodeficiency viruses (SHIVs) encoding biologically relevant clade C envelopes are therefore needed as challenge viruses in vaccine efficacy studies with nonhuman primates. Here we describe the generation of three lineage-related subtype C SHIVs through four successive rapid transfers in rhesus macaques of SHIVC109F.PB4, a molecular clone expressing the soluble-CD4 (sCD4)-sensitive CCR5-tropic clade C envelope of a recently infected subject in Zambia. The viruses differed in their monkey passage histories and neutralization sensitivities but remained R5 tropic. SHIVC109P3 and SHIVC109P3N were recovered from a passage-3 rapid-progressor animal during chronic infection (24 weeks postinfection [wpi]) and at end-stage disease (34 wpi), respectively, and are classified as tier 1B strains, whereas SHIVC109P4 was recovered from a passage-4 normal-progressor macaque at 22 wpi and is a tier 2 virus, more difficult to neutralize. All three viruses were transmitted efficiently via intrarectal inoculation, reaching peak viral loads of 10 7 to 10 9 RNA copies/ml plasma and establishing viremia at various set points. Notably, one of seven (GC98) and two of six (CL31, FI08) SHIVC109P3- and SHIVC109P3N-infected macaques, respectively, progressed to AIDS, with neuropathologies observed in GC98 and FI08, as well as coreceptor switching in the latter. These findings support the use of these new SHIVC109F.PB4-derived viruses to study the immunopathology of HIV-1 clade C infection and to evaluate envelope-based AIDS vaccines in nonhuman primates.

Objective—Development of an effective vaccine or topical compound to prevent HIV transmission remains a major goal for control of the AIDS pandemic. Using a nonhuman primate model of heterosexual HIV-1 transmission, we tested whether a topical microbicide that reduces viral infectivity can potentiate the efficacy of a T-cell-based HIV vaccine. Design—A DNA prime and rAd5 virus boost vaccination strategy was employed, and a topical microbicide against the HIV nucleocapsid protein was used. To rigorously test the combination hypothesis, the vaccine constructs contained only two transgenes and the topical microbicide inhibitor was used at a sub-optimal dose. Vaccinees were exposed in the absence and presence of the topical microbicide to repeated vaginal R5 SHIVSF162P3 challenge at an escalating dose to more closely mimic high-risk exposure of women to HIV. Methods—Infection status was determined by PCR. Antiviral immune responses were evaluated by gp120 ELISA and intracellular cytokine staining. Results—A significant delay in SHIV acquisition (Log-rank test; p=0.0416) was seen only in vaccinated macaques that were repeatedly challenged in the presence of the topical microbicide. Peak acute viremia was lower (Mann-Whitney test; p=0.0387) and viral burden was also reduced (Mann-Whitney test; p=0.0252) in the combination-treated animals. Conclusions—The combined use of a topical microbicide to lower the initial viral seeding/ spread and a T-cell-based vaccine to immunologically contain the early virological events of

We recently described a coreceptor switch in rapid progressor (RP) R5 simian-human immunodeficiency virus SF162P3N (SHIV SF162P3N )-infected rhesus macaques that had high virus replication and undetectable or weak and transient antiviral antibody response (S. H. Ho et al., J. Virol. 81: 8621-8633, 2007; S. H. Ho, N. Trunova, A. Gettie, J. Blanchard, and C. Cheng-Mayer, J. Virol. 82: 5653-5656, 2008; and W. Ren et al., J. Virol. 84: 340-351, 2010). The lack of antibody selective pressure, together with the observation that the emerging X4 variants were neutralization sensitive, suggested that the absence or weakening of the virus-specific humoral immune response could be an environmental factor fostering coreceptor switching in vivo . To test this possibility, we treated four macaques with 50 mg/kg of body weight of the anti-CD20 antibody rituximab every 2 to 3 weeks starting from the week prior to intravenous infection with SHIV SF162P3N for a total of six infusions. Rituximab treatment successfully depleted peripheral and lymphoid CD20 + cells for up to 25 weeks according to flow cytometry and immunohistochemical staining, with partial to full recovery in two of the four treated monkeys thereafter. Three of the four treated macaques failed to mount a detectable anti-SHIV antibody response, while the response was delayed in the remaining animal. The three seronegative macaques progressed to disease, but in none of them could the presence of X4 variants be demonstrated by V3 sequence and tropism analyses. Furthermore, viruses did not evolve early in these diseased macaques to be more soluble CD4 sensitive. These results demonstrate that the absence or diminution of humoral immune responses by itself is insufficient to drive the R5-to-X4 switch and the neutralization susceptibility of the evolving viruses.

Here, we report that the S -acyl-2-mercaptobenzamide thioester (SAMT) class of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NCp7) inhibitors was able to prevent transmission of HIV-1 from infected cells, including primary cells. Furthermore, when SAMTs were introduced during an HIV-1 challenge of cervical explant tissue, inhibition of dissemination of infectious virus by cells emigrating from the tissue explants was observed. Preliminary studies using a rhesus macaque vaginal challenge model with mixed R5 and X4 simian-human immunodeficiency virus infection found that five of six monkeys were completely protected, with the remaining animal being partially protected, infected only by the R5 virus. These data suggest that SAMTs may be promising new drug candidates for further development in anti-HIV-1 topical microbicide applications.

The human immunodeficiency virus (HIV) enters target cells via interaction of the viral glycoprotein with the cellular receptor CD4 and two principal coreceptors, CCR5 (R5 viruses) and CXCR4 (X4 viruses). Most HIV-1 transmissions result in a predominantly R5 virus infection. With time, X4 variants arise and coexist with R5 virus variants in approximately 50% of subtype B infected individuals. The underlying basis for virus coreceptor switch late in infection remains an enigma, but will be important to understand given that the appearance of X4 virus in HIV-1 infected patients inevitably heralds an unfavorable clinical outcome. Recently, emergence of X4 viruses was observed in rhesus macaques experimentally infected with a CCR5-tropic simian-human immunodeficiency virus (SHIV) with progression to disease, providing some insights into the process of coreceptor switching in vivo. Further studies in this animal model should enhance our understanding of the mechanistic basis for, and obstacles to, coreceptor switch.

We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N (SHIV SF162P3N )-infected macaque CA28, supporting the use of this experimental system to examine factors that drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIV CA28NP ) used both CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIV CA28NL ) used CXCR4 almost exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4 usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent mutational pathways for change in the coreceptor preference of the R5 SHIV SF162P3N isolate in vivo.

Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIVSF33A isolate revealed a threshold inoculum for establishment of systemic virus infection and a dose dependency in overall viral burden and CD4+ T cell depletion. While exposure to inoculum size of 1000 or greater 50% tissue infectious dose (TCID50) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID50 of the same virus. The latter was characterized by intermittent detection of low level virus with no evidence of seroconversion or CD4+ T cell decline, but with signs of an ongoing antiviral T cell immune response. Upon vaginal re-challenge with the same limiting dose 11–12 weeks after the first, classic pathogenic X4 SHIVSF33A infection was established in four of the seven previously exposed seronegative macaques, implying enhanced susceptibility to systemic infection with prior exposure. Pre-existing peripheral SIV gag-specific CD4+ T cells were more readily demonstrable in macaques that became systemically infected following re-exposure than those that were not. In contrast, early presence of circulating polyfunctional cytokine secreting CD8+ T cells or strong virus-specific proliferative responses in draining lymph nodes and in the gut associated lymphoid tissue (GALT) following the first exposure was associated with protection from systemic re-infection. These studies identify the gut and lymphoid tissues proximal to the genital tract as sites of robust CD8 T lymphocyte responses that contribute to containment of virus spread following vaginal transmission.

The basis for the switch from CCR5 to CXCR4 coreceptor usage seen in ∼50% of human immunodeficiency virus type 1 (HIV-1) subtype B-infected individuals as disease advances is not well understood. Among the reasons proposed are target cell limitation and better immune recognition of the CXCR4 (X4)-tropic compared to the CCR5 (R5)-tropic virus. We document here X4 virus emergence in a rhesus macaque (RM) infected with R5-tropic simian/human immunodeficiency virus, demonstrating that coreceptor switch can happen in a nonhuman primate model of HIV/AIDS. The switch to CXCR4 usage in RM requires envelope sequence changes in the V3 loop that are similar to those found in humans, suggesting that the R5-to-X4 evolution pathways in the two hosts overlap. Interestingly, compared to the inoculating R5 virus, the emerging CXCR4-using virus is highly neutralization sensitive. This finding, coupled with the observation of X4 evolution and appearance in an animal with undetectable circulating virus-specific antibody and low cellular immune responses, lends further support to an inhibitory role of antiviral immunity in HIV-1 coreceptor switch.

We examined the effect of inoculum dose on SHIV transmission and infection. We found that repeated low-dose intravaginal exposure with either R5-SHIVSF162P3 or X4-SHIVSF33A results in infections that are blunted and rapidly controlled. Interestingly, although the transmission rate after all repeated exposures is comparable for the two viruses, the probability of low-dose vaginal transmission is greater for the X4 than R5 virus. Furthermore, X4-SHIVSF33A replication predominates in low-dose dually-exposed macaques, suggesting that it is better at establishing a systemic infection following transmission. However, X4-SHIVSF33A advantage in transmission and infection is not observed in macaques inoculated intravenously with low-dose mixed inoculum. The finding that although matched in tissue culture infectious dose, the X4 inoculum is more complex leads us to hypothesize that the greater genetic heterogeneity of the X4 virus population may have rendered it less susceptible to the severe bottleneck effects imposed by IVAG inoculation with small doses, allowing for greater probability of transmission and establishment of a generalized infection. These data have implications for HIV-1 transmission and infection in humans.

To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIV SF162P3N and 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs. IMPORTANCE HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIV SF162P3N -infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare, but their development might have been faster in some of the studied macaques. Plasma mapping with monomeric gp120 indicated that most bnAbs bind to the envelope trimer rather than the gp120 monomer. In support of this, none of the isolated gp120-reactive monoclonal antibodies (MAbs) displayed the neutralization breadth observed in the corresponding plasma. However, the MAb sequences revealed similarity between human and macaque genes used to encode some V3-directed MAbs. Our study sheds light on the timing and development of bnAbs in SHIV-infected macaques in comparison to HIV-1-infected humans and highlights the use of envelope trimer probes for efficient recovery of bnAbs.

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathioneS-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.

Objectives: To assess the safety and distribution of a cellulose acetate 1,2-benzene-dicarboxylate (CAP) gel formulation in rhesus macaques as part of the development process for its use as a vaginally administered product in humans. Design: The similarities between the reproductive physiology, anatomy and vaginal microflora of human and non-human primates makes non-human primates a relevant animal model to assess the safety and distribution of candidate anti-HIV microbicides. Methods: CAP gel was instilled once or once daily for 4 days into the vaginal vault of rhesus macaques. Colposcopy and magnetic resonance imaging were performed to detect adverse effects and spread of CAP, respectively. Additionally, vaginal pH and composition of the vaginal micorflora in macaques before, during and after CAP instillations were determined, and vaginal biopsies obtained following repeated CAP exposures were examined to further document its safety. Results: CAP is safe for repeated use and exhibits a favorable distribution profile, showing no evidence of penetration into cells that line the vaginal epithelium. Further, the presence of CAP has no adverse effect on vaginal pH or the composition of the vaginal microflora, and does not induce vaginal epithelial thinning or inflammation. Conclusions: CAP gel shows minimal toxicity in vivo, supporting its use as a candidate vaginal microbicide in humans.

Objectives: To evaluate the protective efficacy of cellulose acetate 1,2-benzenedicarboxylate (CAP) formulated in a glycerol-based gel against infection with CXCR4 (X4) and CCR5 (R5) viruses in the simian/human immunodeficiency virus (SHIV)/rhesus macaque model of HIV-1 transmission. Design: Mucosal infection of non-human primates is a reasonable model for use in the investigation of HIV-1 intervention strategies. Methods: Rhesus macaques treated with Depo-Provera 5 weeks prior to challenge were inoculated intravaginally twice, over a period of 6 h with mixed inocula of pathogenic X4- and R5-SHIV in the presence or absence of CAP. Plasma viral load, peripheral and mucosal CD4 T cell counts as well as the genotype of the circulating virus were determined. Results: CAP protected seven of ten macaques against transmission of both X4- and R5-SHIV, reaching statistically significant values (P= 0.0256). Delayed and/or reduced virus replication, as well as blunting of peripheral and mucosal CD4 T cell loss was noted in the three macaques that were infected in the CAP treated group compared to those in the placebo group. Further, protection conferred by CAP appeared to be more effective against X4- than R5-SHIV infection. Conclusions: CAP is protective against highly permissive challenges with X4 and R5 viruses in vivo. Research on further development of this promising compound as a candidate microbicide for the prevention of sexual HIV-1 transmission is therefore warranted.

Multiple mechanisms are used by the hu- man immunodeficiency virus type 1 (HIV-1) to inter- fere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV- infected primary macrophages remains unclear. Cur- rently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic greenfluorescent protein (GFP)-tagged HIV-1 viruses, which express the com- plete viral genome, and used these to assess the expres- sion of human leukocyte antigen (HLA)-A2 on the sur- face of productively infected macrophages. The re- porter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte- derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analy- ses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frame- shift or a single point mutation in Nef at prolines 74 P and P 80 . The impact of Nef on HLA-A2 surface expres- sion in MDM was also confirmed by confocal micros- copy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages. J. Leukoc. Biol. 78: 675-685; 2005.

It is well documented that removal of the V1V2 region or of the V2 loop alone from the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) increases the susceptibility of these viruses to neutralization by antibodies. The specific role of the V1 loop in defining the neutralization susceptibility of HIV is, however, not well documented. Our current studies indicate that although the V1V2 region is a global modulator of the HIV-1 neutralization susceptibility, the individual roles the V1 and V2 loops have in defining the neutralization susceptibility profile of HIV-1 differ and in some cases are opposite. While deletion of the V2 loop renders the virus more susceptible to neutralization by antibodies that recognize diverse epitopes, in particular certain ones located in the CD4 binding site and the V3 loop, deletion of the V1 loop renders the virus refractory to neutralization, especially by antibodies that recognize CD4-induced epitopes and certain CD4-site binding antibodies. Our current studies also indicate that the relative involvement of the V2 loop of the HIV-1 envelope during virus-cell entry appears to be envelope background dependent. As a result, although deletion of the V2 loop from the clade B, R5-tropic SF162 HIV-1 virus resulted in a virus that was replication competent, the same modification introduced on the background of two other R5-tropic isolates, SF128A (clade B) or SF170 (clade A), abrogated the ability of these envelopes to mediate virus-cell entry.

hTid-1, a human homolog of the Drosophila tumor suppressor l(2)Tid and a novel DnaJ protein, regulates the activity of nuclear factor kappaB (NF-kappaB), but its mechanism is not established. We report here that hTid-1 strongly associated with the cytoplasmic protein complex of NF-kappaB-IkappaB through direct interaction with IkappaBalpha/beta and the IKKalpha/beta subunits of the IkappaB kinase complex. These interactions resulted in suppression of the IKK activity in a J-domain-dependent fashion and led to the cytoplasmic retention and enhanced stability of IkappaB. Overexpression of hTid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status. Adherent cultured cells transduced with Ad.hTid-1 detached from the dish surface. Morphological changes consistent with apoptosis and cell death were evident 48 h after Ad.EGFP-hTid-1 transduction. In contrast, cells transduced with Ad.EGFP or Ad.EGFP-hTd-1DeltaN100, a mutant that has the N-terminal J domain deletion and that lost suppressive activity on IKK, continued to proliferate. Similar data were obtained with A375 human melanoma cells. Ad.EGFP or Ad.EGFP-hTd-1DeltaN100 ex vivo-transduced A375 cells injected subcutaneously into nude mice produced growing tumors, whereas Ad.EGFP-hTid-1-transduced cells did not. Collectively, the data suggest that hTid-1 represses the activity of NF-kappaB through physical and functional interactions with the IKK complex and IkappaB and, in doing so, it modulates cell growth and death.

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1SF162, were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

Newborn rhesus macaques were infected with two chimeric simian-human immunodeficiency virus (SHIV) strains which contain unique human immunodeficiency virus type 1 (HIV-1)envgenes and exhibit distinct phenotypes. Infection with either the CCR5-specific SHIVSF162P3or the CXCR4-utilizing SHIVSF33Aresulted in clinical manifestations consistent with simian AIDS. Most prominent in this study was the detection of severe thymic involution in all SHIVSF33A-infected infants, which is very similar to HIV-1-induced thymic dysfunction in children who exhibit a rapid pattern of disease progression. In contrast, SHIVSF162P3induced only a minor disruption in thymic morphology. Consistent with the distribution of the coreceptors CXCR4 and CCR5 within the thymus, the expression of SHIVSF162P3was restricted to the thymic medulla, whereas SHIVSF33Awas preferentially detected in the cortex. This dichotomy of tissue tropism is similar to the differential tropism of HIV-1 isolates observed in the reconstituted human thymus in SCID-hu mice. Accordingly, our results show that the SHIV-monkey model can be used for the molecular dissection of cell and tissue tropisms controlled by the HIV-1envgene and for the analysis of mechanisms of viral immunopathogenesis in AIDS. Furthermore, these findings could help explain the rapid progression of disease observed in some HIV-1-infected children.

Sooty mangabeys (Cercocebus atys) showed age-dependent changes in T cell regeneration. Younger animals had a high percentage of CD4+ CD45RA + T cells and a high concentration of T cell receptor excisional circles (TRECs) in peripheral blood, which indicated active thymopoiesis. In contrast, older animals had an increased T cell turnover, which suggested that most T cell production occurred in the periphery. In addition, the number of peripheral CD4+ T cells naturally decreased with age. Non-pathogenic SIVsm infection did not significantly change the T cell proliferation rate or the TREC concentration, though it did cause a moderate loss of peripheral CD4 + T cells. The viral load correlated negatively with age, which could be accounted for by the reduced availability of CD4 + target cells in older mangabeys. Thus, the number of susceptible target cells may be a limiting factor in natural SIV infection.

We coinfected rhesus macaques with CXC chemokine receptor 4- and CC chemokine receptor 5-specific simian/human immunodeficiency viruses (SHIVs) to elucidate the basis for the early dominance of R5-tropic strains seen in HIV-infected humans. We found no intrinsic barrier to the transmission and dissemination of high-dose X4-SHIV in the dually infected macaques. In animals that maintained a viral set point, the R5 virus predominated. The time of appearance of R5 dominance coincided with the development of virus-specific immunity (3–6 weeks postinfection), suggestive of differential immune control of the two viruses. Indeed, after depletion of CD8 + T cells in the coinfected animals, X4 virus emerged, supporting the concept that differential CD8 + T cell-mediated immune control of X4- and R5-SHIV replication is responsible for the selective outgrowth of R5 viruses. These findings provide critical insights into a key question related to HIV pathogenesis and have important implications for the development and testing of antiviral vaccines and therapeutics.

In vivo passage of non-pathogenic, CCR5-tropic simian/human immunodeficiency virus (SHIV) - SHIVsf162 resulted in a pathogenic isolate, SHIVsf162p3. In an attempt to characterize envelope (Env)-mediated properties that may contribute to its pathogenicity, major (P3 major) and minor (P3 minor) Env gp120 variants were cloned from the plasma of a SHIVsf162p3-infected animal, and expressed in the context of luciferase reporter viruses. Entry mediated by these envelopes and susceptibility to neutralization by CD4 induced-site (CD4i) antibodies (MAbs) was analyzed in comparison to parental SF162. Sequence analysis revealed that the P3 major and minor variant Envs contained 14 and 17 amino acid changes, respectively, compared with SF162. The rank order of entry mediated by the three envelopes was P3 major > SF162 > P3 minor, whereas the reverse order was observed for susceptibility to neutralization by CD4i MAbs. Since CD4i epitopes overlap the coreceptor (CoR) binding site, these findings suggest that the amino acid changes accumulated upon in vivo passage of SHIVsf162 result in Env gp120 structural rearrangements that modulate the exposure and/or conformation of the CoR binding site. This, in turn, led to increased entry and infectivity of the P3 major variant and may be responsible, in part, for the enhanced pathogenicity of SHIVsf162p3.

HIV pseudotypes bearing native hepatitis C virus (HCV) glycoproteins (strain H and Con1) are infectious for the human hepatoma cell lines Huh-7 and PLC/PR5. Infectivity depends on coexpression of both E1 and E2 glycoproteins, is pH-dependent, and can be neutralized by mAbs mapping to amino acids 412–447 within E2. Cell-surface expression of one or all of the candidate receptor molecules (CD81, low-density lipoprotein receptor, scavenger receptor class B type 1, and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin) failed to confer permissivity to HIV–HCV pseudotype infection. However, HIV–HCV pseudotype infectivity was inhibited by a recombinant soluble form of CD81 and a mAb specific for CD81, suggesting that CD81 may be a component of a receptor complex.

In vivo studies for understanding viral transmission and replication, host immune responses, and pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection would greatly benefit from the establishment of a small-animal model. In this study, we explored the potential of American mink ( Mustera vison ) as a susceptible host. We found that primary cells and cell lines derived from this species efficiently supported trans -activation of the HIV-1 long terminal repeat by Tat. Accordingly, the cysteine residue at position 261, which has been shown to be important for interaction of the human cyclin T1 with the HIV-1 regulatory protein Tat, is conserved in the mink homologue. No species-specific defect in Rev function could be detected in mink cells. In addition, primary splenocytes, fibroblasts, and the Mv.1.Lu cell line from American mink supported early as well as late HIV-1 gene expression following infection with vesicular stomatitis G protein-pseudotyped HIV-1 viruses, at levels comparable to those seen with permissive human cells. Furthermore, the mink Mv.1.Lu cell line stably expressing human CD4 and CCR5 receptors supported a spreading HIV-1 infection with few, if any, deficiencies compared to findings in human cell lines. This indicates the potential of HIV-1 to replicate in these cells once the blockade at the stage of virus entry has been removed. These results clearly show that cells from American mink generally pose no functional intracellular block to HIV-1 replication, and collectively they raise the possibility that this animal species could be engineered to support HIV-1 infection, providing a useful small-animal model for evaluating de novo infection by HIV-1.

The live, attenuated vaccine simian immunodeficiency virus SIVmac239Δnef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4 + T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in the nef gene, resulting in a frameshift that restored the original nef open reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4 + T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncated nef gene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Δ2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Δnef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Δ2nef was similar to that of SIVmac239Δnef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Δ2nef replicated more efficiently than SIVmac239Δnef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Δ2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing the nef deletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Δ2 abolished the replicative advantage in PBMCs, demonstrating that the Nef-Δ2 protein, rather than the structure of the U3 region of the LTR, was responsible for the increase in viral replication. Taken together, these results show that SIV with a deletion in the nef gene can revert to virulence and that expression of a form of nef with multiple deletions may contribute to this process by increasing viral replication.

Long-acting GSK1265744 (GSK744 LA) is a strand transfer inhibitor of the HIV/SIV (simian immunodeficiency virus) integrase and was shown to be an effective preexposure prophylaxis (PrEP) agent in a low-dose intrarectal SHIV (simian-human immunodeficiency virus) rhesus macaque challenge model. We examined the pharmacokinetics and efficacy of GSK744 LA as PrEP against repeat high-dose intravaginal SHIV challenge in female rhesus macaques treated with Depo-Provera (depot medroxyprogesterone acetate), which promotes viral transmission vaginally. When Depo-Provera-treated female rhesus macaques were dosed with GSK744 LA (50 mg/kg) monthly, systemic and tissue drug concentrations were lower than previously observed in male rhesus macaques. GSK744 concentrations were fivefold lower on average in cervical tissues than in rectal tissues. Eight female rhesus macaques were treated with GSK744 LA at week 0, and four female rhesus macaques served as controls. All animals received a high-dose challenge of SHIV162P3 at week 1. No infection was detected in GSK744 LA-treated rhesus macaques, whereas viremia was detected 1 to 2 weeks after SHIV challenge in all control animals. The GSK744 LA-treated rhesus macaques were given a second administration of drug at week 4 and further challenged at weeks 5 and 7. GSK744 LA treatment protected six of eight female rhesus macaques against three high-dose SHIV challenges, whereas all control animals became infected after the first challenge (P = 0.0003, log-rank test). These results support further clinical development of GSK744 LA for PrEP.

The potential role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) binding in human immunodeficiency virus transmission across the mucosal barrier was investigated by assessing the ability of simian-human immunodeficiency chimeric viruses (SHIVs) showing varying degrees of mucosal transmissibility to bind the DC-SIGN expressed on the surface of transfected cells. We found that gp120 of the highly transmissible, pathogenic CCR5-tropic SHIVSF162P3bound human and rhesus DC-SIGN with an efficiency threefold or greater than that of gp120 of the nonpathogenic, poorly transmissible parental SHIVSF162, and this increase in binding to the DC-SIGN of the SHIVSF162P3envelope gp120 translated into an enhancement of T-cell infection intrans. The presence of an additional glycan at the N-terminal base of the V2 loop of SHIVSF162P3gp120 compared to that of the parental virus was shown to be responsible for the increase in binding to DC-SIGN. Interestingly, this glycan also conferred escape from autologous neutralization, raising the possibility that the modification occurred as a result of immune selection. Our data suggest that more-efficient binding of envelope gp120 to DC-SIGN could be relevant to the enhanced mucosal transmissibility of SHIVSF162P3compared to that of parental SHIVSF162.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established model to study acquired immunodeficiency syndrome (AIDS) pathogenesis. Such a controlled system allows for detailedanalysis of the molecular determinants of viral pathogenesis in addition to studying host-specific immune responses that modulate disease progression. Furthermore, the use of a pathogenic molecular clone affords the opportunity to study both viral evolution within a host and to examine the generation of tissue specific variants. In this report we describe viral diversification within tissues of two rhesus macaques infected intravenously with the CXCR4-specific molecular clone SHIV S F 3 3 A 2 . Heteroduplex tracking analysis (HTA) was used to determine the complexity of viral DNA within distinct lymphoid tissues. Not surprising, heterogeneity of the proviral quasispecies in tissues obtained during the acute infection was limited. However, tissues obtained at necropsy harbored a more diverse and often different population of env variants. As the inoculating virus is a molecular clone, the variants generated are likely due to the presence of tissue specific selective forces rather than a founder's effect.

hTid-1, a human DnaJ protein, is a novel cellular target for HTLV-1 Tax. Here, we show that hTid-1 represses NF-kappaB activity induced by Tax as well as other activators such as tumor necrosis factor alpha (TNFalpha) and Bcl10. hTid-1 specifically suppresses serine phosphorylation of IkappaBalpha by activated IkappaB kinase beta (IKKbeta), but the activities of other serine kinases including p38, ERK2, and JNK1 are not affected. The suppressive activity of hTid-1 on IKKbeta requires a functional J domain that mediates association with heat shock proteins and results in prolonging the half-life of the NF-kappaB inhibitors IkappaBalpha and IkappaBbeta. Collectively, our data suggest that hTid-1, in association with heat shock proteins, exerts a negative regulatory effect on the NF-kappaB activity induced by various extracellular and intracellular activators including HTLV-1 Tax.

Simian-human immunodeficiency viruses (SHIVs) are chimeric viruses engineered to test the function or immunogenicity of human immunodeficiency virus (HIV) gene products in simian models. Frequently used constructs contain an HIV type 1 (HIV-1) DNA fragment containing the tat, rev, vpu, and env genes inserted in the proviral genome of SIVmac239, a molecular clone that causes simian AIDS in rhesus macaques (3, 11, 22, 28, 35, 37, 47, 49). SHIVs play a key role in the development of vaccines that are directed against the HIV-1 envelope glycoprotein and are also valuable tools in the understanding of AIDS pathogenesis (41, 42). Comparative studies of SHIVs that use CXCR4 and the CCR5 coreceptor (X4 and R5 SHIVs, respectively) have revealed the role of tropism in the rate and localization of CD4+ T-cell depletion (19). Pathogenic X4 SHIVs typically cause a severe depletion of CD4+ T lymphocytes in blood and peripheral lymph nodes within 1 month of infection (14, 21, 27, 36). Pathogenic isolates of dualtropic R5X4 SHIVs also cause rapid CD4+ T-cell depletion (29, 46, 50), while pathogenic R5 SHIVs cause a more protracted decrease of peripheral CD4+ T cells similar to that seen in SIVmac infection (18). Rhesus macaques chronically infected with pathogenic X4 or R5X4 SHIVs often maintain a high viral load in spite of an almost complete depletion of CD4+ T lymphocytes, a finding that may be explained by the productive infection of activated macrophages (25). SHIV constructs do not readily induce disease in rhesus macaques and require in vivo adaptation and often serial in vivo passages to acquire the capacity to replicate at high levels, deplete CD4+ T cells, and induce simian AIDS. Most pathogenic SHIV isolates, such as SHIVKU-1, SHIV-89.6P, and SHIV162P3, have been recovered after serial transfer of bone marrow or of blood from infected animals (19, 28, 46). Administration of an anti-CD8 monoclonal antibody (MAb) during primary infection with clone SHIVDH-12 resulted in CD4+ T-cell depletion in one rhesus macaque, indicating that immunosuppression could favor the emergence of pathogenic SHIVs (26). SHIVSF33 differs from other isolates in that it evolved into a pathogenic virus without serial in vivo passages or immunosuppressive treatment. The original virus replicated to intermediate levels in rhesus macaques and did not cause CD4+ T-cell depletion, except in one juvenile animal which developed signs of simian AIDS 2 years after inoculation. The isolate recovered from this animal, designated SHIVSF33A (A, adapted), replicated to high titers, induced a rapid and profound depletion of peripheral CD4+ T cells, and caused an AIDS-like syndrome when passaged intravenously in rhesus macaques (36, 37). In addition, SHIVSF33A could be transmitted by the vaginal route even though it used exclusively the CXCR4 coreceptor for entry (21). Comparison of original SHIV constructs with their in vivo-adapted counterparts provides a system to map the viral determinants of SHIV virulence (29, 56). Genetic analyses of pathogenic SHIV molecular clones have demonstrated that virulence is primarily modulated by adaptive changes in the two subunits of the envelope glycoprotein (2, 15, 16, 30, 33). We showed previously that, in the context of the SHIVSF33 genome, replacing the V1-to-V5 region of the env gene with corresponding sequences amplified from SHIVSF33A-infected animals was sufficient to confer pathogenicity (20). The extent of CD4+ T-cell depletion and the disease course in animals infected with recombinant molecular clone SHIVSF33A2 were similar to those caused by the SHIVSF33A isolate, demonstrating that pathogenic determinants mapped to the V1-to-V5 region of gp120. The aim of the present study was to characterize the properties conferred by the envelope glycoprotein of pathogenic SHIVSF33A molecular clones. We found that changes in gp120 were responsible for increases in fusogenicity, cytopathicity, replication capacity, and neutralization resistance of SHIVSF33A and thus conferred a set of properties that could account for the pathogenic phenotype observed in vivo. Furthermore, a conserved CD4-induced epitope that overlaps with the coreceptor binding site was spontaneously exposed at the surfaces of SHIVSF33A virions, even in the absence of CD4 binding. This finding provided a structural basis for the enhanced fusogenicity and replication capacity of pathogenic SHIVSF33A.

Background Severe genetic bottleneck occurs during HIV-1 sexual transmission whereby most infections are initiated by a single transmitted/founder (T/F) virus. Similar observations had been made in nonhuman primates exposed mucosally to SIV/SHIV. We previously reported variable clinical outcome in rhesus macaques inoculated intravaginally (ivg) with a high dose of R5 SHIVSF162P3N. Given the potential contributions of viral diversity to HIV-1 persistence and AIDS pathogenesis and recombination between retroviral genomes increases the genetic diversity, we tested the hypothesis that transmission of multiple variants contributes to heightened levels of virus replication and faster disease progression in the SHIVSF162P3N ivg-infected monkeys. Results We found that the differences in viral replication and disease progression between the transiently viremic (TV; n = 2), chronically-infected (CP; n = 8) and rapid progressor (RP; n = 4) ivg-infected macaques cannot be explained by which variant in the inoculum was infecting the animal. Rather, transmission of a single variant was observed in both TV rhesus, with 1–2 T/F viruses found in the CPs and 2–4 in all four RP macaques. Moreover, the genetic relatedness of the T/F viruses in the CP monkeys with multivariant transmission was greater than that seen in the RPs. Biological characterization of a subset of T/F envelopes from chronic and rapid progressors revealed differences in their ability to mediate entry into monocyte-derived macrophages, with enhanced macrophage tropism observed in the former as compared to the latter. Conclusion Our study supports the tenet that sequence diversity of the infecting virus contributes to higher steady-state levels of HIV-1 virus replication and faster disease progression and highlights the role of macrophage tropism in HIV-1 transmission and persistence.

Keeping HIV at Bay? Preexposure prophylaxis involving daily doses of drugs can, with variable success rates, interrupt HIV transmission for individuals at high risk of acquiring HIV infection. However, one reason for the variability seen in the response to such drugs is a lack of adherence to the prescribed regimen. Andrews et al. (p. 1151 ) formulated a potent integrase inhibitor as a long-acting agent that protected macaques from repeated intrarectal challenges of simian HIV. Decay of plasma levels of drug were associated with increased susceptibility to infection after virus exposure. The drug levels required for a high degree of protection could potentially be achieved with quarterly injections in humans.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established method to study AIDS pathogenesis and is increasingly used to assess the efficacy of vaccine and antiviral candidates. For these reasons, a detailed understanding of those molecular determinants, which confer pathogenic potential to SHIV viruses, should assist in both rational experimental design and interpretation of results. In this report, we describe the development and in vivo characterization of a pathogenic molecular clone, SHIV SF33A2 , which contains an envelope sequence derived from the CXCR4-dependent isolate, HIV-1 SF33 . Proviral DNA, amplified from a rhesus macaque infected with the pathogenic isolate SHIV SF33A , was substituted into the corresponding region of the parental, nonpathogenic SHIV SF33 genome creating the molecular clone SHIV SF33A2 . Coreceptor specificity of SHIV SF33A2 was determined to be CXCR4 specific. Naive rhesus macaques were productively infected after a single exposure to cell-free SHIV SF33A2 by either the intravenous (IV) or intravaginal (IVAG) routes. Animals infected with SHIV SF33A2 suffered a severe loss of peripheral CD4 + T cells and high acute plasma viremia with development of simian AIDS 9 months after inoculation. Sequence analysis identified 25 discreet amino acid changes within the V1-V5 regions of the envelope protein when compared with the nonpathogenic parental virus. These data indicate that domains within the HIV-1 envelope protein are sufficient to define pathogenic potential in the context of the SIVmac239 genome.

We investigated the underlying mechanism by which the highly conserved N-terminal V3 loop glycan of gp120 conferred resistance to neutralization of human immunodeficiency virus type 1 (HIV-1). We find that the presence or absence of this V3 glycan on clade A and B viruses accorded various degrees of susceptibility to neutralization by antibodies to the CD4 binding site, CD4-induced epitopes, and chemokine receptors. Our data suggest that this carbohydrate moiety on gp120 blocks access to the binding site for CD4 and modulates the chemokine receptor binding site of phenotypically diverse clade A and clade B isolates. Its presence also contributes to the masking of CD4-induced epitopes on clade B envelopes. These findings reveal a common mechanism by which diverse HIV-1 isolates escape immune recognition. Furthermore, the observation that conserved functional epitopes of HIV-1 are more exposed on V3 glycan-deficient envelope glycoproteins provides a basis for exploring the use of these envelopes as vaccine components.

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) remain healthy though they harbor viral loads comparable to those in rhesus macaques that progress to AIDS. To assess the immunologic basis of disease resistance in mangabeys, we compared the effect of SIV infection on T-cell regeneration in both monkey species. Measurement of the proliferation marker Ki-67 by flow cytometry showed that mangabeys harbored proliferating T cells at a level of 3 to 4% in peripheral blood irrespective of their infection status. In contrast, rhesus macaques demonstrated a naturally high fraction of proliferating T cells (7%) that increased two- to threefold following SIV infection. Ki-67+T cells were predominantly CD45RA−, indicating increased proliferation of memory cells in macaques. Quantitation of an episomal DNA product of T-cell receptor α rearrangement (termed α1 circle) showed that the concentration of recent thymic emigrants in blood decreased with age over a 2-log unit range in both monkey species, consistent with age-related thymic involution. SIV infection caused a limited decrease of α1 circle numbers in mangabeys as well as in macaques. Dilution of α1 circles by T-cell proliferation likely contributed to this decrease, since α1 circle numbers and Ki-67+fractions correlated negatively. These findings are compatible with immune exhaustion mediated by abnormal T-cell proliferation, rather than with early thymic failure, in SIV-infected macaques. Normal T-cell turnover in SIV-infected mangabeys provides an explanation for the long-term maintenance of a functional immune system in these hosts.

Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication in both primary T lymphocytes and monocyte-derived macrophages. This enhancement phenotype has been linked to the ability of Nef to modulate the activity of cellular kinases. We find that despite the reported high-affinity interaction between Nef and the Src kinase Hck in vitro, a Nef-Hck interaction in the context of HIV-1-infected primary macrophages is not detectable. However, Nef binding and activation of the PAK-related kinase and phosphorylation of its substrate could be readily detected in both infected primary T lymphocytes and macrophages. Furthermore, we show that this substrate is a complex composed of the recently characterized PAK interacting partner PIX (PAK-interacting guanine nucleotide exchange factor) and its tightly associated p95 protein. PAK and PIX-p95 appear to be differentially activated and phosphorylated depending on the intracellular environment in which nef is expressed. These results identify the PIX-p95 complex as a novel effector of Nef in primary cells and suggest that the regulation of the PAK signaling pathway may differ in T cells and macrophages.

The chemokine receptor CCR5 is known to be a critical determinant of human immunodeficiency virus (HIV) transmission and pathogenesis in the human host. Towards the development of a macaque model to evaluate the efficacy of vaccines and therapeutics against infection with CCR5-specific viruses, and to delineate the pathogenic properties of such viruses, we constructed a chimeric simian human immunodeficiency virus, SHIV(SF162), containing the env, tat, rev, and vpu genes from HIV-1(SF162) (R5, MT/NSI) in the context of the molecular clone simian immunodeficiency virus, SIV(mac239). Virus generated from this molecular clone was used to intravenously infect two juvenile macaques, followed by three consecutive serial blood/bone marrow transfusions. Animals infected with parental SHIV(SF162) (P1) had detectable levels of viral replication (as determined by p27(gag) production) within days of infection; however, viral set-points fell below detection by Week 3. Late passage animals (P3 and P4) had a two-log increase in the level of plasma p27(gag) antigen. These results demonstrate that in vivo serial passage of the R5-specific SHIV(SF162) enhanced its replicative capacity.

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIV SF33 , into a pathogenic biologic viral variant, designated SHIV SF33A . In the present study, we show that SHIV SF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIV SF33A ; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIV SF33A , mutant viruses were generated on the backbone of parental SHIV SF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIV SF33 . The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.

Several studies have reported on the differential susceptibility to antibody-mediated neutralization between primary (PR) and laboratory-adapted (LA) human immunodeficiency virus type 1 (HIV-1) isolates (1, 2, 6, 23, 24, 32, 39, 40, 45), the latter being more susceptible than the former to neutralization. To a large extent, this difference is explained by a documented differential exposure of neutralization epitopes within oligomeric envelope structures of PR and LA isolates. Such epitopes are less exposed on PR isolates than LA isolates (1, 24, 36, 40). The identification of the regions that shield neutralization epitopes and the nature of the neutralization epitopes themselves on PR isolates should shed light on the mechanism(s) by which HIV evades neutralization by serum antibodies and greatly benefit the design of effective HIV envelope-based immunogens. It has been reported that simultaneous deletion of the V1 and V2 loops of the neutralization-sensitive LA isolate HIV-1IIIb increases even further its susceptibility to neutralization by certain monoclonal antibodies (MAbs) whose epitopes are located within the V3 loop, the CD4-binding site, and conserved gp120 regions (3). We previously reported that the individual deletion of the V1 (17 amino acids) and V2 (30 amino acids) loops of the PR, neutralization-resistant HIV-1 isolate SF162 (7, 35, 39) does not reduce the potential of the virus to replicate in peripheral blood mononuclear cells (PBMC) or alter the coreceptor function of the envelope protein (38). Immunochemical analysis of the two mutant envelopes revealed that the deleted V1 and V2 loop sequences differentially affect the exposure and conformation of specific gp120 epitopes. While deletion of the V2 loop affects the structure of certain complex epitopes which comprise elements of the CD4-binding site, deletion of the V1 loop alters the conformation and exposure of specific epitopes located in the V2 and V3 loops and the CD4-binding site. However, regardless of these specific structural changes, neither deletion alters the overall ability of the envelope to interact with CD4 or its ability to undergo conformational changes upon receptor binding (38). In this study, we examined whether individual partial deletion of the V1 or V2 loop exposes cryptic neutralization epitopes on the surface of SF162 virions. We show that the V2, but not the V1, loop deletion renders the virus, termed SF162ΔV2, highly susceptible to serum-mediated neutralization. Most importantly, SF162ΔV2 becomes neutralizable by sera obtained from patients infected not only with clade B HIV-1 isolates but also with clade A, C, D, E, and F HIV-1 isolates. The introduced V2 loop deletion does not destabilize the gp120-gp41 subunit association, and the virus remains as resistant to antibody-mediated gp120-virion dissociation as wild-type (wt) SF162. Based on these observations, we suggest that this particular V2 loop deletion unveils an envelope region rich in neutralization epitopes whose overall structure is conserved among PR isolates. Neutralization studies conducted with various anti-gp120 MAbs failed to identify the precise epitopes forming this region, but it appears that it comprises structural elements of the CD4-binding site and conserved regions of gp120.

Infection of macaques with chimeric simian/human immunodeficiency virus (SHIV) expressing the envelope protein of HIV-1 provides a model system for studying HIV-1 infection in humans. To this end, four rhesus macaques (Macaca mulatta) were given a single intravaginal (IVAG) inoculation of cell-free SHIVSF33A and longitudinal samples of peripheral blood and lymph nodes were analyzed for viremia, antigenemia, and various T-cell populations. Rhesus macaques infected IVAG with SHIVSF33A demonstrated a dramatic decrease in the CD4(+) PBMC subset in the initial weeks after viral exposure, a time that corresponded to peak in plasma viremia and antigenemia. Within 4 months of SHIVSF33A inoculation, partial to complete rebound of the CD4(+) PBMC was seen in these animals. Notably, the regeneration of the CD4(+) subset was associated with regeneration of the naive T-cell population and was concordant with clearance of plasma viremia. DNA heteroduplex tracking assays revealed transmission of minor variants within the SHIVSF33A inoculum to the IVAG-inoculated animals. The cell-free SHIVSF33A inoculum as well as virus isolated from animals early after transmission used the chemokine molecule CXCR4 as the primary cellular coreceptor, demonstrating that viruses expressing envelope glycoproteins of the syncytia inducing (SI) phenotype can be transported across the vaginal mucosa. Although none of the animals has yet to develop clinical symptoms of simian AIDS (SAIDS), infectious virus and viral nucleic acids could be persistently isolated from each animal. Furthermore, animals transfused with blood from IVAG-infected macaques drawn 2 weeks after inoculation suffered a more profound and sustained CD4(+) T-cell loss, persistent plasma viremia, and the development of SAIDS in one animal, indicating that IVAG-passaged SHIVSF33A was pathogenic. Taken together, these results establish that a pathogenic CXCR4-utilizing SHIVSF33A species crossed the cervicovaginal mucosa. Different courses of infection in the IVAG versus transfusion animals suggest that host-mediated responses elicited upon transmission across mucosal barriers may serve to limit viral replication and delay disease progression in the IVAG-infected animals.

Neurocognitive disorders such as dementia and cognitive/motor impairments are among the most significant complications associated with human immunodeficiency virus (HIV) infection, especially in aging populations, yet the pathogenesis remains poorly understood. Activated macrophages and microglia in white matter along with the hallmark multinucleated giant cells are prominent features of HIV encephalitis (HIVE) and of several simian immunodeficiency virus (SIV) models. While infected microglia have been demonstrated in HIVE, this feature is not routinely seen in experimental infections in rhesus macaques using SIV or chimeric simian/HIV (SHIV) strains, limiting utility in HIV-1 pathogenesis and treatment studies. Here, 50 rhesus macaques were inoculated with the CCR5 (R5)-tropic SHIVSF162P3N virus by one of three routes: intravenously (n = 9), intrarectally (n = 17), or intravaginally (n = 24). Forty-three monkeys became viremic, 26 developed AIDS, and 7 (7/26, 27 %) developed giant cell SIV encephalitis (SIVE). Rapid progressor phenotype was evident in five of seven (71 %) macaques with SIVE, and expansion to utilize the CXCR4 coreceptor (X4 coreceptor switch) was observed in four out of seven (57 %). SIVE lesions were present in gray and white matter in the cerebrum, cerebellum, thalamus, and brain stem of affected animals. Lesions were composed of virally infected CD68(+), CD163(+), and HLA-DR(+) macrophages accompanied by white matter damage, necrosis, and astroglial and microglial activation. Importantly, microglial infection was observed, which makes R5 SHIVSF162P3N infection of macaques an attractive animal model not only to study transmission and HIVE pathogenesis but also to conduct preclinical evaluation of therapeutic interventions aimed at eradicating HIV-1 from the central nervous system (CNS).

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV- infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic α-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.