Cecilia Cheng-Mayer

Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles

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

Antibody Protects Macaques against Vaginal Challenge with a Pathogenic R5 Simian/Human Immunodeficiency Virus at Serum Levels Giving Complete Neutralization In Vitro

ABSTRACT 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 SHIV162P4. 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.

HIV-1 nef leads to inhibition or activation of T cells depending on its intracellular localization

Nef of primate lentiviruses is required for viremia and progression to AIDS in monkeys. Negative, positive, and no effects of Nef have also been reported on viral replication in cells. To reconcile these observations, we expressed a hybrid CD8-Nef protein in Jurkat cells. Two opposite phenotypes were found, which depended on the intracellular localization of Nef. Expressed in the cytoplasm or on the cell surface, the chimera inhibited or activated early signaling events from the T cell antigen receptor. Activated Jurkat cells died by apoptosis, and only cells with mutated nef genes expressing truncated Nefs survived, which rendered Nef nonfunctional. These mutations paralleled those in other viral strains passaged in vitro. Not only do these positional effects of Nef reconcile diverse phenotypes of Nef and suggest a role for its N-terminal myristylation, but they also explain effects of Nef in HIV infection and progression to AIDS.

Normal T-Cell Turnover in Sooty Mangabeys Harboring Active Simian Immunodeficiency Virus Infection

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

Mucosal Transmission and Induction of Simian AIDS by CCR5-Specific Simian/Human Immunodeficiency Virus SHIV SF162P3

ABSTRACT Nonhuman primate models are increasingly used in the screening of candidate AIDS vaccine and immunization strategies for advancement to large-scale human trials. The predictive value of such macaque studies is largely dependent upon the fidelity of the model system in mimicking human immunodeficiency virus (HIV) type 1 infection in terms of viral transmission, replication, and pathogenesis. Herein, we describe the efficient mucosal transmission of a CCR5-specific chimeric simian/human immunodeficiency virus, SHIVSF162P3. Female rhesus macaques were infected with SHIVSF162P3 after a single atraumatic application to the cervicovaginal mucosa. The disease course of SHIVSF162P3-infected monkeys is similar and as varied as natural HIV infection in terms of viral replication, gradual loss of CD4+ peripheral blood mononuclear cells, and the development of simian AIDS-defining opportunistic infections. The SHIVSF162P3/macaque model should facilitate direct preclinical assessment of HIV vaccine strategies in addition to antiviral compounds directed towards envelope target cell interactions. Furthermore, this controlled model provides the setting to investigate immunologic responses and putative host-specific susceptibility factors that alter viral transmission and subsequent disease progression.

The N-Terminus of Nef from HIV-1/SIV Associates with a Protein Complex Containing Lck and a Serine Kinase

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

Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques

BACKGROUND The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase. RESULTS This study identifies the Nef-associated kinase as a member of the p21-activated kinase (PAK) family of kinases and investigates the role of this Nef-associated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression. CONCLUSIONS Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAK-related kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAK-related kinase.

The V1, V2, and V3 Regions of the Human Immunodeficiency Virus Type 1 Envelope Differentially Affect the Viral Phenotype in an Isolate-Dependent Manner

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

The N-Terminal V3 Loop Glycan Modulates the Interaction of Clade A and B Human Immunodeficiency Virus Type 1 Envelopes with CD4 and Chemokine Receptors

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

The use of nonhuman primate models in HIV vaccine development

Cecilia Morgan and colleagues outline a two-stage nonhuman primate screening strategy for T cell-based HIV-1 vaccines.