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.     

Consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virus-infected macaques

We have analyzed changes to proviral Env gp120 sequences and the development of neutralizing antibodies (NAbs) during 1 year of simian/human immunodeficiency virus SHIV-89.6P infection in 11 Macaca nemestrina macaques. Seven macaques had significant env divergence from that of the inoculum, and macaques with greater divergence had higher titers of homologous NAbs. Substitutions in sequons encoding potential N-linked glycosylation sites (PNGs) were among the first to be established, although overall the total number of sequons did not increase significantly. The majority (19 of 23) of PNGs present in the inoculum were conserved in the sequences from all macaques. Statistically significant variations in PNGs occurred in multiple macaques within constrained regions we term "hot spots," resulting in the selection of sequences more similar to the B consensus. These included additions on V1, the N-terminal side of V4, and the outer region of C2. Complex mutational patterns resulted in convergent PNG shifts in V2 and V5. Charge changes in Env V1V2, resulting in a net acidic charge, and a proline addition in V5 occurred in several macaques. Molecular modeling of the 89.6P sequence showed that the conserved glycans lie on the silent face of Env and that many are proximal to disulfide bonds, while PNG additions and shifts are proximal to the CD4 binding site. Nonsynonymous-to-synonymous substitution ratios suggest that these changes result from selective pressure. This longitudinal and cross-sectional study of mutations in human immunodeficiency virus (HIV) env in the SHIV background provides evidence that there are more constraints on the configuration of the glycan shield than were previously appreciated.     

Factors contributing to spontaneous Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected macaques

Background A cohort of SIV‐infected macaques had been used to investigate the effect of dietary supplement, immune status, SIV/AIDS disease progression and serum micronutrients levels on spontaneous acquisition of Enterocytozoon bieneusi infection in SIV‐infected macaques. Methods Twenty‐four SIV‐infected macaques were randomized into 2 groups. One group received a vitamin/mineral supplementation and a second group received a placebo. Both groups were examined for E. bieneusi infection. Results SIV‐infected macaques were more prone to acquire E. bieneusi with the progression of SIV/AIDS, and the increased shedding of infectious spores was directly associated with decreased CD4 lymphocyte and increased circulating SIV, in both supplemented and unsupplemented groups of animals. Dietary supplementation, body composition factors and serum micronutrients levels however had no association with the acquisition of E. bieneusi infection in these animals. Conclusions Acquisition of E. bieneusi infection is related to SIV disease progression, CD4 counts and viral load but independent of changes in body composition and serum micronutrient levels.     

Comprehensive analysis of nef functions selected in simian immunodeficiency virus-infected macaques

A variety of simian immunodeficiency virus (SIVmac) nef mutants have been investigated to clarify which in vitro Nef functions contribute to efficient viral replication and pathogenicity in rhesus macaques. Most of these nef alleles, however, were only functionally characterized for their ability to down-modulate CD4 and class I major histocompatibility complex (MHC-I) cell surface expression and to enhance SIV replication and infectivity. To obtain information on the in vivo relevance of more recently established Nef functions, we examined the ability of a large panel of constructed SIVmac Nef mutants and of variants that emerged in infected macaques to down-regulate CD3, CD28, and MHC-II and to up-regulate the MHC-II-associated invariant chain (Ii). We found that all these four Nef functions were restored in SIV-infected macaques. In most cases, however, the initial mutations and the changes selected in vivo affected several in vitro Nef functions. For example, truncated Nef proteins that emerged in animals infected with SIVmac239 containing a 152-bp deletion in nef efficiently modulated both CD3 and Ii surface expression. Overall, our results suggest that the effect of Nef on each of the six cellular receptors investigated contributes to viral fitness in the infected host but also indicate that modulation of CD3, MHC-I, MHC-II, or Ii surface expression alone is insufficient for SIV virulence.     

Induction of autoantibodies to CCR5 in macaques and subsequent effects upon challenge with an R5-tropic simian/human immunodeficiency virus

Antibodies against CCR5, the major coreceptor for human immunodeficiency virus type 1 (HIV-1), may have antiviral potential as viral fusion inhibitors. In this study, we generated a virus-like particle (VLP)-based vaccine that effectively breaks B-cell tolerance and elicits autoantibodies against CCR5 in pig-tailed macaques. Initial studies in mice identified a polypeptide comprising the N-terminal domain of pig-tailed macaque CCR5 fused to streptavidin that, when conjugated at high density to bovine papillomavirus major capsid protein L1 VLPs, induced high-titer immunoglobulin G (IgG) that bound to a macaque CCR5-expressing cell line in vitro. In macaques, CCR5 peptide-conjugated VLP preparations induced high-avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV(SF162P3) in vitro. Although the anti-CCR5 IgG titers declined with time, autoantibody levels were boosted upon revaccination. Vaccinated macaques remained healthy for a period of over 3 years after the initial immunization, and no decline in the number of CCR5-expressing T cells was detected. To test the prophylactic efficacy of CCR5 autoantibodies, immunized macaques were challenged with SHIV(SF162P3). Although the plasma-associated virus in half of six control macaques declined to undetectable levels, viral loads were lower, declined more rapidly, and eventually became undetectable in all five macaques in which CCR5 autoantibodies had been elicited. In addition, in the four vaccinated macaques with higher autoantibody titers, viral loads and time to control of viremia were significantly decreased relative to controls, indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.     

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.     

Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies

Passively transferred neutralizing antibodies can block lentivirus infection, but their role in postexposure prophylaxis is poorly understood. In this nonhuman-primate study, the effects of short-term antibody therapy on 5-year disease progression, virus load, and host immunity were explored. We reported previously that postinfection passive treatment with polyclonal immune globulin with high neutralizing titers against SIVsmE660 (SIVIG) significantly improved the 67-week health of SIVsmE660-infected Macaca mulatta macaques. Four of six treated macaques maintained low or undetectable levels of virus in plasma, compared with one of ten controls, while two rapid progressors controlled viremia only as long as the SIVIG was present. SIVIG treatment delayed the de novo production of envelope (Env)-specific antibodies by 8 weeks (13). We show here that differences in disease progression were also significant at 5 years postinfection, excluding rapid progressors (P = 0.05). Macaques that maintained 相似文献   

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Acute and chronic T cell dynamics in the livers of simian immunodeficiency virus-infected macaques

The mucosal immune system, particularly the gastrointestinal tract, is critically involved in the pathogenesis of human immunodeficiency virus (HIV) infection. Since the liver drains most of the substances coming from the intestinal tract, it may also play a role in the pathogenesis of HIV infection. Here we examined the percentages and absolute numbers of T cell subsets in the liver in normal and simian immunodeficiency virus (SIV)-infected macaques. Most of the T cells in the liver were CD8(+) memory cells, and most of these had an effector memory (CD95(+) CD28(-)) phenotype. CD4(+) T cells constituted approximately 20% of the liver T cell population, but the vast majority of these were also memory (CD95(+)) CCR5(+) cells, suggesting they were potential targets for viral infection. After SIV infection, CD4(+) T cells were markedly reduced, and increased proliferation and absolute numbers of CD8(+) T cells were detected in the liver. These data suggest that the liver is a major source of antigenic stimulation for promoting CD8(+) T cells and possibly a source for early CD4(+) T cell infection and destruction.     

Use of inhibitors to evaluate coreceptor usage by simian and simian/human immunodeficiency viruses and human immunodeficiency virus type 2 in primary cells

We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative coreceptor that is expressed in the cells of some individuals. SIV(mac)239 and SIV(mac)251 (from macaques) were also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors. The replication in human PBMC of SIV(rcm) (from a red-capped mangabey), a virus which uses CCR2 but not CCR5 for entry, was blocked by TAK-779, suggesting that CCR2 is indeed the paramount coreceptor for this virus in primary cells.     

Effect of B-cell depletion on coreceptor switching in R5 simian-human immunodeficiency virus infection of rhesus macaques

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.     

Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques.

Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact.     

Antiviral antibodies and T cells are present in the foreskin of simian immunodeficiency virus-infected rhesus macaques

No information exists regarding immune responses to human immunodeficiency virus (HIV) infection in the foreskin or glans of the human penis, although this is a key tissue for HIV transmission. To address this gap, we characterized antiviral immune responses in foreskin of male rhesus macaques (RMs) inoculated with simian immunodeficiency virus (SIV) strain SIVmac251 by penile foreskin exposure. We found a complete population of immune cells in the foreskin and glans of normal RMs, although B cells were less common than CD4(+) and CD8(+) T cells. IgG-secreting cells were detected by enzyme-linked immunospot (ELISPOT) assay in cell suspensions made from the foreskin. In the foreskin and glans of SIV-infected RMs, although B cells were less common than CD4(+) and CD8(+) T cells, SIV-specific IgG antibody was present in foreskin secretions. In addition, cytokine-secreting SIV-specific CD8(+) T cells were readily found in cell suspensions made from the foreskin. Although potential HIV target cells were found in and under the epithelium covering all penile surfaces, the presence of antiviral effector B and T cells in the foreskin suggests that vaccines may be able to elicit immunity in this critical site to protect men from acquiring HIV.     

Lymphocyte activation during acute simian/human immunodeficiency virus SHIV(89.6PD) infection in macaques

Host-virus interactions control disease progression in human immunodeficiency virus-infected human beings and in nonhuman primates infected with simian or simian/human immunodeficiency viruses (SHIV). These interactions evolve rapidly during acute infection and are key to the mechanisms of viral persistence and AIDS. SHIV(89.6PD) infection in rhesus macaques can deplete CD4(+) T cells from the peripheral blood, spleen, and lymph nodes within 2 weeks after exposure and is a model for virulent, acute infection. Lymphocytes isolated from blood and tissues during the interval of acute SHIV(89.6PD) infection have lost the capacity to proliferate in response to phytohemagglutinin (PHA). T-cell unresponsiveness to mitogen occurred within 1 week after mucosal inoculation yet prior to massive CD4(+) T-cell depletion and extensive virus dissemination. The lack of mitogen response was due to apoptosis in vitro, and increased activation marker expression on circulating T cells in vivo coincided with the appearance of PHA-induced apoptosis in vitro. Inappropriately high immune stimulation associated with rapid loss of mature CD4(+) T cells suggested that activation-induced cell death is a mechanism for helper T-cell depletion in the brief period before widespread virus dissemination. Elevated levels of lymphocyte activation likely enhance SHIV(89.6PD) replication, thus increasing the loss of CD4(+) T cells and diminishing the levels of virus-specific immunity that remain after acute infection. The level of surviving immunity may dictate the capacity to control virus replication and disease progression. We describe this level of immune competence as the host set point to show its pivotal role in AIDS pathogenesis.     

V3 loop-determined coreceptor preference dictates the dynamics of CD4+-T-cell loss in simian-human immunodeficiency virus-infected macaques

We used experimental infection of rhesus macaques with envelope gp120 V3 loop isogenic simian-human immunodeficiency virus (SHIV) molecular clones to more clearly define the impact of human immunodeficiency virus type 1 coreceptor usage in target cell selectivity and the rates of CD4+-T-cell depletion. Functional assays demonstrate that substitution of the V3 loop of the pathogenic CXCR4-tropic (X4) SHIV(SF33A2) molecular clone with the corresponding sequences from the CCR5-tropic (R5) SHIV(SF162P3) isolate resulted in a switch of coreceptor usage from CXCR4 to CCR5. The resultant R5 clone, designated SHIV(SF33A2(V3)), is replication competent in vivo, infecting two of two macaques by intravenous inoculation with peak viremia that is comparable to that seen in monkeys infected with X4-SHIV(SF33A2). But while primary infection with the X4 clone was accompanied by rapid and significant loss of peripheral and secondary lymphoid CD4+ T lymphocytes, infection with R5-SHIV(SF33A2(V3)) led to only a modest and transient loss. However, substantial depletion of intestinal CD4+ T cells was observed in R5-SHIV(SF33A2(V3))-infected macaques. Moreover, na?ve T cells that expressed high levels of CXCR4 were rapidly depleted in X4-SHIV(SF33A2)-infected macaques, whereas R5-SHIV(SF33A2(V3)) infection mainly affected memory T cells that expressed CCR5. These findings in a unique isogenic system illustrate that coreceptor usage is the principal determinant of tissue and target cell specificity of the virus in vivo and dictates the dynamics of CD4+-T-cell depletion during SHIV infection.     

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.     

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.     

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.     

Different mutational pathways to CXCR4 coreceptor switch of CCR5-using simian-human immunodeficiency virus

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.