Michael W. Cho

N-Linked Glycosylation Sites Adjacent to and within the V1/V2 and the V3 Loops of Dualtropic Human Immunodeficiency Virus Type 1 Isolate DH12 gp120 Affect Coreceptor Usage and Cellular Tropism

ABSTRACT The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

Polyvalent Envelope Glycoprotein Vaccine Elicits a Broader Neutralizing Antibody Response but Is Unable To Provide Sterilizing Protection against Heterologous Simian/Human Immunodeficiency Virus Infection in Pigtailed Macaques

ABSTRACT The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIVDH12) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIVDH12 strongly correlated with the level of NAbs directed against the virus on the day of challenge (P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIVDH12 challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

Human Immunodeficiency Virus Type 1 Envelope gp120-Induced Partial T-Cell Receptor Signaling Creates an F-Actin-Depleted Zone in the Virological Synapse

ABSTRACT Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) occurs via a virological synapse (VS), a tight cell-cell junction formed between HIV-infected cells and target cells in which the HIV-1-infected cell polarizes and releases virions toward the noninfected target cell in a gp120- and intercellular adhesion molecule 1 (ICAM-1)-dependent process. The response of the target cell has been less studied. We utilized supported planar bilayers presenting gp120 and ICAM-1 as a reductionist model for the infected-cell membrane and investigated its effect on the target CD4 T cell. This study shows that HIV-1 gp120 interaction with its receptors is initially organized into microclusters that undergo F-actin-dependent consolidation into a central supramolecular activation complex (cSMAC). Src kinases are active in both gp120 microclusters and in the VS cSMAC. The early T-cell receptor (TCR) signaling machinery is partially activated at the VS, and signaling does not propagate to trigger Ca2+ elevation or increase CD69 expression. However, these partial TCR signals act locally to create an F-actin-depleted zone. We propose a model in which the F-actin-depleted zone formed within the target CD4 T cell enhances the reception of virions by releasing the physical barrier for HIV-1 entry and facilitating postentry events.

Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein

Abstract The etiological agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus SARS-CoV. Similar to other coronaviruses, spike (S)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, S-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. To begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. Our results show that the protein has an electrophoretic mobility of about 160–170 kDa. The protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kDa of the apparent protein mass. The detection of S-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. We were able to pseudotype murine leukemia virus particles with S-protein and produce SARS pseudoviruses. Pseudoviruses infected Vero E6 cells in a pH-independent manner and the infection could be specifically inhibited by convalescent sera. Consistent with low levels of antibodies against S-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. To facilitate quantifying pseudovirus-infected cells, which are stained blue with X-Gal, we devised an automated procedure using an ELISPOT analyzer. The high-throughput capacity of this procedure and the safety of using SARS pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against SARS-CoV.

Human Immunodeficiency Virus Type 1 Envelope gp120 Induces a Stop Signal and Virological Synapse Formation in Noninfected CD4+ T Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4+ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4+ T cells.

Control of Viremia and Prevention of Simian-Human Immunodeficiency Virus-Induced Disease in Rhesus Macaques Immunized with Recombinant Vaccinia Viruses plus Inactivated Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Particles

ABSTRACT An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4+ T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4+ T cells, and have remained healthy for more than 15 months postinfection. CD8+ T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4+ T-cell loss following its inoculation into a naïve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus.

Structural Characterization of HIV gp41 with the Membrane-proximal External Region

Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.

Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor

Abstract Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus, SARS-CoV. Virus entry into cells is mediated through interactions between spike (S) glycoprotein and angiotensin-converting enzyme 2 (ACE2). Alanine scanning mutagenesis analysis was performed to identify determinants on ACE2 critical for SARS-CoV infection. Results indicated that charged amino acids between residues 22 and 57 were important, K26 and D30, in particular. Peptides representing various regions of ACE2 critical for virus infection were chemically synthesized and evaluated for antiviral activity. Two peptides (a.a. 22–44 and 22–57) exhibited a modest antiviral activity with IC50 of about 50 μM and 6 μM, respectively. One peptide comprised of two discontinuous segments of ACE2 (a.a. 22–44 and 351–357) artificially linked together by glycine, exhibited a potent antiviral activity with IC50 of about 0.1 μM. This novel peptide is a promising candidate as a therapeutic agent against this deadly emerging pathogen.

Identification of gp120 Regions Targeted by a Highly Potent Neutralizing Antiserum Elicited in a Chimpanzee Inoculated with a Primary Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1DH12) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1DH12, nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1–V2 and V4 regions of HIV-1DH12 could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.

Development of a Safe and Rapid Neutralization Assay Using Murine Leukemia Virus Pseudotyped with HIV Type 1 Envelope Glycoprotein Lacking the Cytoplasmic Domain

Neutralizing antibody (NAb) is a critical component of an immune system that can potentially provide sterilizing protection against human immunodeficiency virus type 1 (HIV-1). Therefore, an in vitro assay that can rapidly, safely, and accurately evaluate the NAb response vaccine candidates elicit, especially against a large number of HIV-1 variants, would be highly valuable. It has been demonstrated that HIV-1 envelope glycoprotein lacking the cytoplasmic domain can pseudotype murine leukemia virus encoding the beta-galactosidase gene and that this pseudovirus can specifically infect CD4(+) cells (Schnierle BS, Stitz J, Bosch V, et al.: Proc Natl Acad Sci USA 1997;94:8640-8645). Because the pseudovirus is not biohazardous and because the infection can be quantitatively determined within 2 days, we examined the feasibility of using the pseudovirus for high-throughput neutralization assays for HIV-1. We have generated viruses pseudotyped with gp140 of six different HIV-1 isolates (LAI, RF, Bal, AD8, 89.6, and DH12). All six pseudoviruses were infectious and exhibited expected coreceptor usage phenotype in HOS-CD4 cells expressing either CCR5 or CXCR4. More importantly, the neutralization sensitivity profile of these pseudoviruses was virtually identical to that observed from more conventional neutralization assays using either HIV-1 or SHIV. All pseudoviruses could be neutralized by broadly reactive human monoclonal antibody IgG1 b12. Our results indicate that the pseudoviruses are ideal for high-throughput evaluation of immune sera for their capacity to broadly neutralize a large number of HIV-1 isolates.