CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 nef protein in pediatric AIDS progression

The human immunodeficiency virus type 1 (HIV-1) nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived nef alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived nef alleles, compared to nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary nef alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.     

Impact of Nef-mediated downregulation of major histocompatibility complex class I on immune response to simian immunodeficiency virus

Functional activities that have been ascribed to the nef gene product of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) include CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, downregulation of other plasma membrane proteins, and lymphocyte activation. Monkeys were infected experimentally with SIV containing difficult-to-revert mutations in nef that selectively eliminated MHC downregulation but not these other activities. Monkeys infected with these mutant forms of SIV exhibited higher levels of CD8(+) T-cell responses 4 to 16 weeks postinfection than seen in monkeys infected with the parental wild-type virus. Furthermore, unusual compensatory mutations appeared by 16 to 32 weeks postinfection which restored some or all of the MHC-downregulating activity. These results indicate that nef does serve to limit the virus-specific CD8 cellular response of the host and that the ability to downregulate MHC class I contributes importantly to the totality of nef function.     

Pol-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells prior to Nef-mediated major histocompatibility complex class I downregulation

Effective, vaccine-induced CD8+ T-cell responses should recognize infected cells early enough to prevent production of progeny virions. We have recently shown that Gag-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells at 2 h postinfection, whereas Env-specific CD8+ T cells do not recognize infected cells until much later in infection. However, it remains unknown when other proteins present in the viral particle are presented to CD8+ T cells after infection. To address this issue, we explored CD8+ T-cell recognition of epitopes derived from two other relatively large virion proteins, Pol and Nef. Surprisingly, infected cells efficiently presented CD8+ T-cell epitopes from virion-derived Pol proteins within 2 h of infection. In contrast, Nef-specific CD8+ T cells did not recognize infected cells until 12 h postinfection. Additionally, we show that SIVmac239 Nef downregulated surface major histocompatibility complex class I (MHC-I) molecules beginning at 12 h postinfection, concomitant with presentation of Nef-derived CD8+ T-cell epitopes. Finally, Pol-specific CD8+ T cells eliminated infected cells as early as 6 h postinfection, well before MHC-I downregulation, suggesting a previously underappreciated antiviral role for Pol-specific CD8+ T cells.     

Human immunodeficiency virus type 1 Vpu protein interacts with CD74 and modulates major histocompatibility complex class II presentation

The human immunodeficiency virus type 1 (HIV-1) Vpu accessory protein is a transmembrane protein that down regulates CD4 expression and promotes the release of new virions. We screened a human leukocyte-specific yeast two-hybrid expression library to discover novel Vpu-interacting cellular proteins. The major histocompatibility complex class II (MHC II) invariant chain, also called Ii or CD74, was found to be one such protein. We show direct binding of Vpu and CD74 by using a yeast two-hybrid assay and coimmunoprecipitation from HIV-1-infected cells. The cytoplasmic region of Vpu was found to interact with the 30-amino-acid cytoplasmic tail of CD74. Human monocytic U937 cells infected with wild-type or Vpu-defective HIV-1 and transfected cells showed that Vpu down modulated the surface expression of mature MHC II molecules. The reduction in cell surface mature MHC II molecules correlated with decreased antigen presentation to T cells in culture. Thus, the Vpu protein also contributes to viral persistence by attenuating immune responses during HIV infection. This report further exemplifies the rich diversity and redundancy shown by HIV in immune evasion.     

CD4 coexpression regulates DC-SIGN-mediated transmission of human immunodeficiency virus type 1

Dendritic cells (DCs) potently stimulate the cell-cell transmission of human immunodeficiency virus type 1 (HIV-1). However, the mechanisms that underlie DC transmission of HIV-1 to CD4⁺ T cells are not fully understood. DC-SIGN, a C-type lectin, efficiently promotes HIV-1 trans infection. DC-SIGN is expressed in monocyte-derived DCs (MDDCs), macrophage subsets, activated B lymphocytes, and various mucosal tissues. MDDC-mediated HIV-1 transmission to CD4⁺ T cells involves DC-SIGN-dependent and -independent mechanisms. DC-SIGN transmission of HIV-1 depends on the donor cell type. HIV-1 Nef can upregulate DC-SIGN expression and promote DC-T-cell clustering and HIV-1 spread. Nef also downregulates CD4 expression; however, the effect of the CD4 downmodulation on DC-mediated HIV-1 transmission has not been examined. Here, we report that CD4 expression levels correlate with inefficient HIV-1 transmission by monocytic cells expressing DC-SIGN. Expression of CD4 on Raji B cells strongly impaired DC-SIGN-mediated HIV-1 transmission to T cells. By contrast, enhanced HIV-1 transmission was observed when CD4 molecules on MDDCs and DC-SIGN-CD4-expressing cell lines were blocked with specific antibodies. Coexpression of CD4 and DC-SIGN in Raji cells promoted the internalization and intracellular retention of HIV-1. Interestingly, internalized HIV-1 particles were sorted and confined to late endosomal compartments that were positive for CD63 and CD81. Furthermore, in HIV-1-infected MDDCs, significant downregulation of CD4 by Nef expression correlated with enhanced viral transmission. These results suggest that CD4, which is present at various levels in DC-SIGN-positive primary cells, is a key regulator of HIV-1 transmission.     

HIV Nef-mediated major histocompatibility complex class I down-modulation is independent of Arf6 activity

HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.     

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.     

Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.     

Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit

Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the mu but not the gamma/sigma subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical mu-binding motif. These data identify the mu subunit of AP-1 (mu1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with mu1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based mu-binding signal in the MHC-I CD by AP-1.     

Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.     

Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins.     

Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.     

Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis

The nef gene products encoded by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus type 1 (SIV-1) increase viral loads in infected hosts and accelerate clinical progression to AIDS. Nef exhibits a spectrum of biological activities, including the ability to downregulate surface expression of CD4 and major histocompatibility complex (MHC) class I antigens, to alter the state of T-cell activation, and to enhance the infectivity of viral particles. To determine which of these in vitro functions most closely correlates with the pathogenic effects of Nef in vivo, we constructed recombinant HIV-1 NL4-3 viruses carrying mutations within the nef gene that selectively impair these functions. These mutant viruses were evaluated for pathogenic potential in severe combined immunodeficiency (SCID) mice implanted with human fetal thymus and liver (SCID-hu Thy/Liv mice), in which virus-mediated depletion of thymocytes is known to be Nef dependent. Disruption of the polyproline type II helix (Pxx)4 within Nef (required for binding of Hck and p21-activated kinase-like kinases, downregulation of MHC class I, and enhancement of HIV-1 infectivity in vitro but dispensable for CD4 downregulation) did not impair thymocyte depletion in virus-infected Thy/Liv human thymus implants. Conversely, three separate point mutations in Nef that compromised its ability to downregulate CD4 attenuated thymocyte depletion while not diminishing viral replication. These findings indicate that the functional ability of Nef to downregulate CD4 and not MHC class I downregulation, Hck or PAK binding, or (Pxx)4-associated enhancement of infectivity most closely correlates with Nef-mediated enhancement of HIV-1 pathogenicity in vivo. Nef-mediated CD4 downregulation merits consideration as a new target for the development of small-molecule inhibitors.     

Analysis of pigtail macaque major histocompatibility complex class I molecules presenting immunodominant simian immunodeficiency virus epitopes

Successful human immunodeficiency virus (HIV) vaccines will need to induce effective T-cell immunity. We studied immunodominant simian immunodeficiency virus (SIV) Gag-specific T-cell responses and their restricting major histocompatibility complex (MHC) class I alleles in pigtail macaques (Macaca nemestrina), an increasingly common primate model for the study of HIV infection of humans. CD8+ T-cell responses to an SIV epitope, Gag164-172KP9, were present in at least 15 of 36 outbred pigtail macaques. The immunodominant KP9-specific response accounted for the majority (mean, 63%) of the SIV Gag response. Sequencing from six macaques identified 7 new Mane-A and 13 new Mane-B MHC class I alleles. One new allele, Mane-A*10, was common to four macaques that responded to the KP9 epitope. We adapted reference strand-mediated conformational analysis (RSCA) to MHC class I genotype M. nemestrina. Mane-A*10 was detected in macaques presenting KP9 studied by RSCA but was absent from non-KP9-presenting macaques. Expressed on class I-deficient cells, Mane-A*10, but not other pigtail macaque MHC class I molecules, efficiently presented KP9 to responder T cells, confirming that Mane-A*10 restricts the KP9 epitope. Importantly, naive pigtail macaques infected with SIVmac251 that respond to KP9 had significantly reduced plasma SIV viral levels (log10 0.87 copies/ml; P=0.025) compared to those of macaques not responding to KP9. The identification of this common M. nemestrina MHC class I allele restricting a functionally important immunodominant SIV Gag epitope establishes a basis for studying CD8+ T-cell responses against AIDS in an important, widely available nonhuman primate species.     

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.     

Differential effects of primary human immunodeficiency virus type 1 nef sequences on downregulation of CD4 and MHC class I

We analyzed two primary nef sequences, KS2 (subtype B) and K306 (subtype D), each directly isolated from patients. Cell lines constitutively expressing respective Nef proteins were constructed using a retroviral vector. There were significant differences in the ability to downregulate surface CD4 and MHC class I proteins between different nef sequences. When the nef sequence from NL432 was used as a reference, KS2 Nef demonstrated the highest ability to downregulate MHC class I, whereas it appeared to lack the ability to downregulate CD4. On the contrary, Nef from K306 decreased the level of surface CD4 to a greater extent, but was less effective on downregulation of MHC class I. These results showed that the levels of downregulation of CD4 and MHC class I could significantly vary among HIV strains and that two well-known functions of Nef, downregulation of CD4 and MHC class I, would be separated.     

CD8+ and CD20+ lymphocytes cooperate to control acute simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys: modulation by major histocompatibility complex genotype

We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIV(DH12R-Clone 7), like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+ T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIV(DH12R-Clone 8) experience only partial and transient losses of CD4+ T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+ and CD20+ lymphocytes in suppressing the replication of the attenuated SHIV(DH12R-Clone 8) and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+ or CD20+ cells during the SHIV(DH12R-Clone 8) acute infection resulted in the rapid, complete, and irreversible loss of CD4+ T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+ cells; SHIV(DH12R-Clone 8) was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+ and CD20+ B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.     

Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4(+) T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4(+) T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201(+) rhesus monkeys failed to demonstrate tetramer-binding CD4(+) T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4(+) T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201(+) monkeys vaccinated with a recombinant canarypox virus-HIV-1 env construct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4(+) T cells. Thus, Env p46-specific CD4(+) T cells can be detected by tetrameric Mamu-DR*W201-p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.