Janet M. Harouse

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.

Increased Mucosal Transmission but Not Enhanced Pathogenicity of the CCR5-Tropic, Simian AIDS-Inducing Simian/Human Immunodeficiency Virus SHIVSF162P3 Maps to Envelope gp120

ABSTRACT Through rapid serial transfer in vivo, the chimeric CCR5-tropic simian/human immunodeficiency virus SHIVSF162 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 SHIVSF162P3, we cloned and sequenced the dominant envelope gene (encoding P3 gp120) and characterized its functions in vitro. Chimeric SHIVSF162 virus expressing P3 gp120 of the pathogenic variant, designated SHIVSF162PC, was also constructed and assessed for its pathogenicity and mucosal transmissibility in vivo. We found that, compared to wild-type SHIVSF162 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, SHIVSF162PC infected two of two and two of three rhesus macaques by the intravenous and intravaginal routes, respectively. Nevertheless, although peak viremia reached 106 to 107 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 SHIVSF162P3, the determinant(s) of its increased virulence may require additional sequence changes in env gp41 and/or maps to other viral genes.

CD8+ T cell-mediated CXC chemokine receptor 4-simian/human immunodeficiency virus suppression in dually infected rhesus macaques

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

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.

ABSTRACT 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 envconstruct 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.

Entry of Human Immunodeficiency Virus‐1 Into Glial Cells Proceeds via an Alternate, Efficient Pathway

Although the CD4 molecule is the cellular receptor for human immunodeficiency virus‐1 (HIV‐1) in cells of the lymphocyte/monocyte lineage, a number of investigators have also been able to infect cells, including several of central nervous system (CNS) origin, that do not express CD4 protein or mRNA. These infections are generally nonpermissive. To ascertain whether the nonpermissive nature of infection in glial cells is due to an inefficient entry pathway, we prepared a permanently transfected U373‐MG cell line expressing the CD4 molecule and demonstrated that HIV‐1 still replicates at a low level. Furthermore, a virus uptake assay indicated that HIV‐1 enters glial cells effectively, even in the absence of CD4. These results demonstrate that HIV‐1 entry is efficient and that the restrictive nature of the infection in glial cells is due to postentry mechanisms. In addition, these findings support the existence of an alternate, efficient, entry pathway in some glial cells.

A CCR5-tropic simian-HIV molecular clone capable of inducing AIDS in rhesus macaques

We previously reported the derivation of a CCR5 (R5)-tropic pathogenic strain SHIVSF162P3. Here, we show that a simian-HIV (SHIV) molecular clone expressing the entire env gp160 of SHIVSF162P3, termed SHIV P3gp160, could fully recapitulate the in vivo replicative characteristics of the parental isolate. SHIV P3gp160 is mucosally transmissible, preferentially depletes memory CD4+ T cells, and induced simian AIDS in 2 of 6 infected macaques. The availability of an infectious R5 SHIV molecular clone that can be transmitted mucosally and causes disease provides an important reagent for studies of lentiviral pathogenesis and AIDS vaccine research.

Pathogenic determinants of the mucosally transmissible CXCR4-specific SHIV(SF33A2) map to env region.

Summary: Infection of rhesus macaques with chimeric simian‐human immunodeficiency viruses (SHIV) is an established method to study AIDS pathogenesis and is increasingly used to assess the efficacy of vaccine and antiviral candidates. For these reasons, a detailed understanding of those molecular determinants, which confer pathogenic potential to SHIV viruses, should assist in both rational experimental design and interpretation of results. In this report, we describe the development and in vivo characterization of a pathogenic molecular clone, SHIVSF33A2, which contains an envelope sequence derived from the CXCR4‐dependent isolate, HIV‐1SF33. Proviral DNA, amplified from a rhesus macaque infected with the pathogenic isolate SHIVSF33A, was substituted into the corresponding region of the parental, nonpathogenic SHIVSF33 genome creating the molecular clone SHIVSF33A2. Coreceptor specificity of SHIVSF33A2 was determined to be CXCR4 specific. Naive rhesus macaques were productively infected after a single exposure to cell‐free SHIVSF33A2 by either the intravenous (IV) or intravaginal (IVAG) routes. Animals infected with SHIVSF33A2 suffered a severe loss of peripheral CD4+ T cells and high acute plasma viremia with development of simian AIDS 9 months after inoculation. Sequence analysis identified 25 discreet amino acid changes within the V1‐V5 regions of the envelope protein when compared with the nonpathogenic parental virus. These data indicate that domains within the HIV‐1 envelope protein are sufficient to define pathogenic potential in the context of the SIVmac239 genome.

Genetic determinants controlling HIV-1 tropism for CD4−/GalCer+ human intestinal epithelial cells

It is now well-established that some viruses, including adenovirus and Herpes simplex virus, can recognize more than one receptor. The human immunodeficiency virus type 1 (HIV-1) recognizes both the CD4 glycoprotein on the surface of CD4+ lymphocytes and macrophages, and the glycosphingolipid galactosylceramide (GalCer) on cells of neural and intestinal origin. The infection of the CD4−/GalCer+ HT-29 intestinal epithelial cell line is restricted to a subset of T-cell-line-tropic isolates. The determinants responsible for HIV-1 infection of HT-29 cells have been characterized by functional analysis of chimeric proviral clones derived from T-cell-line-tropic (HXB2) and macrophage-tropic isolates (ADA, YU2, and 89.6, respectively). Replacement of the HXB2 V3-loop sequence with that derived from either ADA or YU2 resulted in a virus that could no longer infect HT-29 cells. However, the reciprocal replacement of ADA or YU2 V3-loop by the corresponding HXB2 sequence did not confer the ability to infect HT-29 cells. By contrast, insertion in the 89.6-sequence of a 193-amino-acid fragment from the envelope region of HXB2 containing the V3, V4 and V5 regions resulted in a virus able to infect HT-29. Moreover, recombinant viruses that separate this 193-amino-acid fragment into two distinct chimeras were each able to confer the infection phenotype. Taken together, these data emphasize the importance of the V3 loop in the tropism of HIV-1 for CD4−/GalCer+ intestinal cells. In addition, the conformation of the V3 loop that is operative for GalCer recognition may be modulated by distinct domains of the gp120 molecule.