Coreceptor switch in R5-tropic simian/human immunodeficiency virus-infected macaques

The basis for the switch from CCR5 to CXCR4 coreceptor usage seen in approximately 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.     

Induction of CD8+ cells able to suppress CCR5-tropic simian immunodeficiency virus SIVmac239 replication by controlled infection of CXCR4-tropic simian-human immunodeficiency virus in vaccinated rhesus macaques

Recent recombinant viral vector-based AIDS vaccine trials inducing cellular immune responses have shown control of CXCR4-tropic simian-human immunodeficiency virus (SHIV) replication but difficulty in containment of pathogenic CCR5-tropic simian immunodeficiency virus (SIV) in rhesus macaques. In contrast, controlled infection of live attenuated SIV/SHIV can confer the ability to contain SIV superchallenge in macaques. The specific immune responses responsible for this control may be induced by live virus infection but not consistently by viral vector vaccination, although those responses have not been determined. Here, we have examined in vitro anti-SIV efficacy of CD8+ cells in rhesus macaques that showed prophylactic viral vector vaccine-based control of CXCR4-tropic SHIV89.6PD replication. Analysis of the effect of CD8+ cells obtained at several time points from these macaques on CCR5-tropic SIVmac239 replication in vitro revealed that CD8+ cells in the chronic phase after SHIV challenge suppressed SIV replication more efficiently than those before challenge. SIVmac239 superchallenge of two of these macaques at 3 or 4 years post-SHIV challenge was contained, and the following anti-CD8 antibody administration resulted in transient CD8+ T-cell depletion and appearance of plasma SIVmac239 viremia in both of them. Our results indicate that CD8+ cells acquired the ability to efficiently suppress SIV replication by controlled SHIV infection, suggesting the contribution of CD8+ cell responses induced by controlled live virus infection to containment of HIV/SIV superinfection.     

Enhanced infectivity of an R5-tropic simian/human immunodeficiency virus carrying human immunodeficiency virus type 1 subtype C envelope after serial passages in pig-tailed macaques (Macaca nemestrina)

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.     

Macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV) develop broadly reactive anti-HIV neutralizing antibodies

The development of anti-human immunodeficiency virus (anti-HIV) neutralizing antibodies and the evolution of the viral envelope glycoprotein were monitored in rhesus macaques infected with a CCR5-tropic simian/human immunodeficiency virus (SHIV), SHIVSF162P4. Homologous neutralizing antibodies developed within the first month of infection in the majority of animals, and their titers were independent of the extent and duration of viral replication during chronic infection. The appearance of homologous neutralizing antibody responses was preceded by the appearance of amino acid changes in specific variable and conserved regions of gp120. Amino acid changes first appeared in the V1, V2, C2, and V3 regions and subsequently in the C3, V4, and V5 regions. Heterologous neutralizing antibody responses developed over time only in animals with sustained plasma viremia. Within 2 years postinfection the breadth of these responses was as broad as that observed in certain patients infected with HIV type 1 (HIV-1) for over a decade. Despite the development of broad anti-HIV-1 neutralizing antibody responses, viral replication persisted in these animals due to viral escape. Our studies indicate that cross-reactive neutralizing antibodies are elicited in a subset of SHIVSF162P4 infected macaques and that their development requires continuous viral replication for extended periods of time. More importantly, their late appearance does not prevent progression to disease. The availability of an animal model where cross-reactive anti-HIV neutralizing antibodies are developed may facilitate the identification of virologic and immunologic factors conducive to the development of such antibodies.     

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.     

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.     

Pathogenicity and mucosal transmissibility of the R5-tropic simian/human immunodeficiency virus SHIV(AD8) in rhesus macaques: implications for use in vaccine studies

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.     

Estimating the infectivity of CCR5-tropic simian immunodeficiency virus SIV(mac251) in the gut

CD4+ T-cell depletion during acute human immunodeficiency virus infection occurs predominantly in the gastrointestinal mucosa. Using experimental data on SIV(mac251) viral load in blood and CD4+ T cells in the jejunum, we modeled the kinetics of CD4+ T-cell infection and death and estimated the viral infectivity. The infectivity of SIV(mac251) is higher than previously estimated for SHIV89.6P infection, but this higher infectivity is offset by a lower average peak viral load in SIV(mac251). Thus, the dynamics of target cell infection and death are remarkably similar between a CXCR4- and a CCR5-tropic infection in vivo.     

Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs

Aminooxypentane (AOP)-RANTES efficiently and specifically blocks entry of non-syncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) into host cells. Inhibition appears to be mediated by increased intracellular retention of the CCR5 coreceptor- AOP-RANTES complex and/or competitive binding of AOP-RANTES with NSI R5 HIV-1 isolates for CCR5. Although AOP-RANTES and other beta-chemokine analogs are potent inhibitors, the extreme heterogeneity of the HIV-1 envelope glycoproteins (gp120 and gp41) and variable coreceptor usage may affect the susceptibility of variant HIV-1 strains to these drugs. Using the same peripheral blood mononuclear cells (PBMC) with all isolates, we observed a significant variation in AOP-RANTES inhibition of 13 primary NSI R5 isolates; 50% inhibitory concentrations (IC(50)) ranged from 0.04 nM with HIV-1(A-92RW009) to 1.3 nM with HIV-1(B-BaL). Experiments performed on the same isolate (HIV-1(B-BaL)) with PBMC from different donors revealed no isolate-specific variation in AOP-RANTES IC(50) values but did show a considerable difference in virus replication efficiency. Exclusive entry via the CCR5 coreceptor by these NSI R5 isolates suggests that variable inhibition by AOP-RANTES is not due to alternative coreceptor usage but rather differential CCR5 binding. Analysis of the envelope V3 loop sequence linked a threonine or arginine at position 319 (numbering based on the HXB2 genome) with AOP-RANTES resistance. With the exception of one isolate, A319 was associated with increased sensitivity to AOP-RANTES inhibition. Distribution of AOP-RANTES IC(50) values with these isolates has promoted ongoing screens for new CCR5 agonists that show broad inhibition of HIV-1 variants.     

Persistence of pathogenic challenge virus in macaques protected by simian immunodeficiency virus SIVmacDeltanef

Live attenuated simian immunodeficiency virus (SIV) is the most efficient vaccine yet developed in monkey models of human immunodeficiency virus infection. In all successful vaccine trials, attenuation was achieved by inactivating at least the nef gene. We investigated some virological and immunological characteristics of five rhesus macaques immunized with a nef-inactivated SIVmac251 molecular clone (SIVmac251Deltanef) and challenged 15 months later with the pathogenic SIVmac251 isolate. Three animals were killed 2 weeks postchallenge (p.c.) to search for the challenge virus and to assess immunological changes in various organs. The other two animals have been monitored up for 7 years p.c., with clinical and nef gene changes being noted. The animals killed showed no increase in viral load and no sign of a secondary immune response, although the challenged virus was occasionally detected by PCR. In one of the monkeys being monitored, the vaccine virus persisted and an additional deletion occurred in nef. In the other monkey that was monitored, the challenge and the vaccine (Deltanef) viruses were both detected by PCR until a virus with a hybrid nef allele was isolated 48 months p.c. This nef hybrid encodes a 245-amino-acid protein. Thus, our results show (i) that monkeys were not totally protected against homologous virus challenge but controlled the challenge very efficiently in the absence of a secondary immune response, and (ii) that the challenge and vaccine viruses may persist in a replication-competent form for long periods after the challenge, possibly resulting in recombination between the two viruses.     

High beta-chemokine expression levels in lymphoid tissues of simian/human immunodeficiency virus 89.6-vaccinated rhesus macaques are associated with uncontrolled replication of simian immunodeficiency virus challenge inoculum

Viral suppression by noncytolytic CD8+ T cells, in addition to that by classic antiviral CD8+ cytotoxic T lymphocytes, has been described for human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. However, the role of soluble effector molecules, especially beta-chemokines, in antiviral immunity is still controversial. In an attenuated vaccine model, approximately 60% of animals immunized with simian/human immunodeficiency virus (SHIV) 89.6 and then challenged intravaginally with SIVmac239 controlled viral replication (viral RNA level in plasma, <10(4) copies/ml) and were considered protected (K. Abel, L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller, J. Virol. 77:3099-3118, 2003). To determine the in vivo importance of beta-chemokine secretion and CD8+-T-cell proliferation in the control of viral replication in this vaccine model, we examined the relationship between viral RNA levels in the axillary and genital lymph nodes of vaccinated, protected (n = 20) and vaccinated, unprotected (n = 11) monkeys by measuring beta-chemokine mRNA levels and protein expression, the frequency of CD8+ T cells expressing beta-chemokines, and the extent of CD8+-T-cell proliferation. Tissues from uninfected (n = 3) and unvaccinated, SIVmac239-infected (n = 9) monkeys served as controls. Axillary and genital lymph nodes from unvaccinated and vaccinated, unprotected monkeys had significantly higher beta-chemokine mRNA expression levels and increased numbers of beta-chemokine-positive cells than did vaccinated, protected animals. Furthermore, the lymph nodes of vaccinated, unprotected monkeys had significantly higher numbers of beta-chemokine(+) CD8+ T cells than did vaccinated, protected monkeys. Lymph nodes from vaccinated, unprotected animals also had significantly more CD8+-T-cell proliferation and marked lymph node hyperplasia than the lymph nodes of vaccinated, protected monkeys. Thus, higher levels of virus replication were associated with increased beta-chemokine secretion and there is no evidence that beta-chemokines contributed to the SHIV89.6-mediated control of viral replication after intravaginal challenge with SIVmac239.     

Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239.

Nontraumatic vaginal inoculation of rhesus macaques with a simian/human immunodeficiency virus (SIV/HIV) chimera containing the envelope gene from HIV-1 89.6 (SHIV 89.6) results in systemic infection (Y. Lu, B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045-3050, 1996). A total of five rhesus macaques have each been infected by exposure to at least three intravaginal inoculations of SHIV 89.6. The SHIV 89.6 infection is characterized by a transient viremia that evokes humoral and cellular immune responses to HIV and SIV antigens, but disease does not develop in animals infected with SHIV 89.6. To determine if a previous infection with SHIV 89.6 by vaginal inoculation could protect animals from vaginal challenge with pathogenic SIV, all five animals were intravaginally inoculated twice with pathogenic SIV-mac239. After challenge, all of the SHIV-immunized animals had low or undetectable viral RNA levels in plasma compared to control animals. Three of the five of the SHIV-immunized animals remained virus isolation negative for more than 8 months, while two became virus isolation positive. The presence of SIV Gag-specific cytotoxic T lymphocytes in peripheral blood mononuclear cells and SIV-specific antibodies in cervicovaginal secretions at the time of challenge was associated with resistance to pathogenic SIV infection after vaginal challenge. These results suggest that protection from sexual transmission of HIV may be possible by effectively stimulating both humoral and cellular antiviral immunity in the systemic and genital mucosal immune compartments.     

Increased mucosal transmission but not enhanced pathogenicity of the CCR5-tropic,simian AIDS-inducing simian/human immunodeficiency virus SHIV(SF162P3) maps to envelope gp120

Through rapid serial transfer in vivo, the chimeric CCR5-tropic simian/human immunodeficiency virus SHIV(SF162) evolved from a virus that is nonpathogenic and poorly transmissible across the vaginal mucosa to a variant that still maintains CCR5 usage but which is now pathogenic and establishes intravaginal infection efficiently. To determine whether envelope glycoprotein gp120 is responsible for increased pathogenesis and transmissibility of the variant SHIV(SF162P3), we cloned and sequenced the dominant envelope gene (encoding P3 gp120) and characterized its functions in vitro. Chimeric SHIV(SF162) virus expressing P3 gp120 of the pathogenic variant, designated SHIV(SF162PC), was also constructed and assessed for its pathogenicity and mucosal transmissibility in vivo. We found that, compared to wild-type SHIV(SF162) gp120, P3 gp120 conferred in vitro neutralization resistance and increased entry efficiency of the virus but was compromised in its fusion-inducing capacity. In vivo, SHIV(SF162PC) infected two of two and two of three rhesus macaques by the intravenous and intravaginal routes, respectively. Nevertheless, although peak viremia reached 10(6) to 10(7) RNA copies per ml of plasma in some infected animals and was associated with depletion of gut-associated CD4(+) lymphocytes, none of the animals maintained a viral set point that would be predictive of progression to disease. Together, the data from this study suggest a lack of correlation between entry efficiency and cytopathic properties of envelope glycoproteins with viral pathogenicity. Furthermore, whereas env gp120 contains the determinant for enhanced mucosal transmissibility of SHIV(SF162P3), the determinant(s) of its increased virulence may require additional sequence changes in env gp41 and/or maps to other viral genes.     

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4⁺ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4⁺ CCR5⁺ T cells, as this subset of memory/activated CD4⁺ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4⁺ CCR5⁺ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4⁺ CCR5⁺ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4⁺ T-cell function, as measured by CD4⁺ T-cell count, the fraction of memory CD4⁺ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4⁺ CCR5⁺ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4⁺ CCR5⁺ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4⁺ CCR5⁺ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4⁺ CCR5⁺ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.     

Molecular evolution analysis of the human immunodeficiency virus type 1 envelope in simian/human immunodeficiency virus-infected macaques: implications for challenge dose selection

Since the demonstration that almost 80% of human immunodeficiency virus type 1 (HIV-1) infections result from the transmission of a single variant from the donor, biological features similar to those of HIV mucosal transmission have been reported for macaques inoculated with simian immunodeficiency virus (SIV). Here we describe the early diversification events and the impact of challenge doses on viral kinetics and on the number of variants transmitted in macaques infected with the chimeric simian/human immunodeficiency virus SHIV(sf162p4). We show that there is a correlation between the dose administered and the number of variants transmitted and that certain inoculum variants are preferentially transmitted. This could provide insight into the viral determinants of transmission and could aid in vaccine development. Challenge through the mucosal route with high doses results in the transmission of multiple variants in all the animals. Such an unrealistic scenario could underestimate potential intervention measures. We thus propose the use of molecular evolution analysis to aid in the determination of challenge doses that better mimic the transmission dynamics seen in natural HIV-1 infection.     

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1(Bx08). Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1(Bx08) were detected in animals that received (i) coinoculations with DNA(Bx08) and VLP(Bx08), (ii) DNA(Bx08) followed by ALVAC(Bx08) boosting, and (iii) VLP(Bx08) alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-gamma)-producing cells. These cellular immune responses required the inclusion of DNA(Bx08) in the immunization modality, since few or no IFN-gamma-producing cells were detected in animals that received either VLP(Bx08) or ALVAC(Bx08) alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.     

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV_KU. Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4⁺ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4⁺ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV_KU and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.     

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV(KU). Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4+ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4+ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV(KU) and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.     

Inoculation of baboons and macaques with simian immunodeficiency virus/Mne, a primate lentivirus closely related to human immunodeficiency virus type 2

A primate lymphotropic lentivirus was isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma. This isolate, originally designated M. nemestrina immunodeficiency virus (MnIV) and now classified as simian immunodeficiency virus (SIV/Mne), was inoculated intravenously into three juvenile rhesus monkeys (Macaca mulatta), three juvenile pig-tailed macaques (M. nemestrina), and two juvenile baboons (Papio cynocephalus). All six macaques became viremic by 3 weeks after inoculation, whereas neither of the baboons developed viremia. One pig-tailed macaque died at 15 weeks with suppurative peritonitis secondary to ulcerative, necrotizing colitis. Immunologic abnormalities included a marked decrease in CD4+ peripheral blood lymphocytes. Although five macaques mounted an antibody response to SIV/Mne, the animal that died at 15 weeks remained antibody negative. Three other macaques (two rhesus and one pig-tailed) died 66 to 87 weeks after inoculation after exhibiting progressive weight loss, anemia, and diarrhea. Histopathologic findings at necropsy included various manifestations of immune deficiency, nephropathy, subacute encephalitis, pancreatitis, adenocarcinoma, and lymphoid atrophy. SIV/Mne could be readily isolated from the spleens and lymph nodes of all necropsied macaques, and from the cerebrospinal fluid, brains, bone marrow, livers, and pancreas of some of the animals. SIV antigens were localized by avidin-biotin immunohistochemistry to pancreatic islet cells and to bone marrow endothelial cells. The data suggest that African baboons may be resistant to infection by SIV/Mne, whereas Asian macaques are susceptible to infection with this pathogenic primate lentivirus.     

Suppression of human and simian immunodeficiency virus replication with the CCR5-specific antibody Leronlimab in two species

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1.Trial Registration: ClinicalTrials.gov Identifiers: {"type":"clinical-trial","attrs":{"text":"NCT02175680","term_id":"NCT02175680"}}NCT02175680 and {"type":"clinical-trial","attrs":{"text":"NCT02355184","term_id":"NCT02355184"}}NCT02355184.