Carol D. Weiss

Dissection of Human Immunodeficiency Virus Type 1 Entry with Neutralizing Antibodies to gp41 Fusion Intermediates

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5°C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37°C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37°C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37°C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37°C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.

Peptides Trap the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Fusion Intermediate at Two Sites

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into target cells requires folding of two heptad-repeat regions (N-HR and C-HR) of gp41 into a trimer of N-HR and C-HR hairpins, which brings viral and target cell membranes together to facilitate membrane fusion. Peptides corresponding to the N-HR and C-HR of gp41 are potent inhibitors of HIV infection. Here we report new findings on the mechanism of inhibition of a N-HR peptide and compare these data with inhibition by a C-HR peptide. Using intact envelope glycoprotein (Env) under fusogenic conditions, we show that the N-HR peptide preferentially binds receptor-activated Env and that CD4 binding is sufficient for triggering conformational changes that allow the peptide to bind Env, results similar to those seen with the C-HR peptide. However, activation by both CD4 and chemokine receptors further enhances Env binding by both peptides. We also show that a nonconservative mutation in the N-HR of gp41 abolishes C-HR peptide but not N-HR peptide binding to gp41. These results indicate that there are two distinct sites in receptor-activated Env that are potential targets for drug or vaccine development.

Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: Implications for fusion-inducing conformational changes

ABSTRACT We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5 binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We also demonstrate conformational changes in the C heptad of gp41.

Establishment of retroviral pseudotypes with influenza hemagglutinins from H1, H3, and H5 subtypes for sensitive and specific detection of neutralizing antibodies

Pseudotype reporter viruses provide a safe, quantitative, and high-throughput tool for assessing antibody neutralization for many viruses, including high pathogenicity H5 and H7 influenza A strains. However, adapting this system to other influenza subtypes has been hampered by variations in the protease cleavage site of hemagglutinin (HA) that make it less susceptible to the cleavage required for infectivity. In this report several proteases, reporter vectors, and cell substrates were evaluated while optimizing pseudovirus production, and robust methods were established for sensitive and specific neutralization of pseudotypes carrying influenza H1, H3, and H5 subtype HA that correlates well with titers obtained in microneutralization assays involving replicating influenza virus These findings should facilitate broad use of HA-pseudotypes that remove the need for infectious virus in a range of applications, including neutralization assays for serological surveys of viral infection and evaluations of vaccine sera.

Peptides Corresponding to the Heptad Repeat Motifs in the Transmembrane Protein (gp41) of Human Immunodeficiency Virus Type 1 Elicit Antibodies to Receptor-Activated Conformations of the Envelope Glycoprotein

ABSTRACT Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.

Structure-Function Studies of the Self-Assembly Domain of the Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41

ABSTRACT The coiled-coil region of the human immunodeficiency virus type 1 transmembrane protein (gp41) makes up the interior core of the six-helix bundle structure of the gp41 self-assembly domain. We extended our previous study of this domain (Y. Weng and C. D. Weiss, J. Virol. 72:9676–9682, 1998) by analyzing 23 additional mutants at positions that lie at the interface of the interior core and outer helices. We found nine new functional mutants. For most mutants, the activity could be explained by the ability of the modeled mutants to stabilize the six-helix bundle structure. The present study provides insights into the envelope glycoprotein fusion mechanism and information for rational drug and vaccine design.

Studies of HIV-1 envelope glycoprotein-mediated fusion using a simple fluorescence assay.

ObjectiveTo study HIV envelope glycoprotein (Env)-mediated entry using a sensitive fusion assay. Design and methodsCD4+ lymphocytes or T-cell lines were labelled with fluorescent cytoplasm or membrane markers. Fusion with Env-expressing adherent cells was monitored by observing dye transfer from CD4+ cells to Env cells. ResultsCell-cell fusion began 20–30 min after co-cultivation at 37°C. Pre-binding at 4°C was observed not to decrease the lag phase before fusion. Cells expressing envelope glycoproteins from non-syncytium-inducing (NSI) HIV strains showed dye transfer between two cells without progression to syncytia. A glycosylphosphatidylinositol anchored Env was found to be incapable of mediating membrane fusion, as measured either by lipid or cytoplasm contents mixing. Primary mouse cells expressing human CD4 and mouse 3T3 cells stably expressing both human CD4 and human CD26 did not support fusion with our Env-expressing cells. ConclusionsEnv-mediated cell-cell fusion is a relatively slow process, probably reflecting a multi-step process occurring after CD4 binding and requiring the trans-membrane domain of gp41. Env proteins are able to mediate cell-cell fusion at least under some experimental conditions, indicating that lack of a syncytia phenotype does not rule out the possibility of fusion occurring between only two or a few cells.

Cross-Neutralizing Antibodies to Pandemic 2009 H1N1 and Recent Seasonal H1N1 Influenza A Strains Influenced by a Mutation in Hemagglutinin Subunit 2

Pandemic 2009 H1N1 influenza A virus (2009 H1N1) differs from H1N1 strains that circulated in the past 50 years, but resembles the A/New Jersey/1976 H1N1 strain used in the 1976 swine influenza vaccine. We investigated whether sera from persons immunized with the 1976 swine influenza or recent seasonal influenza vaccines, or both, neutralize 2009 H1N1. Using retroviral pseudovirions bearing hemagglutinins on their surface (HA-pseudotypes), we found that 77% of the sera collected in 1976 after immunization with the A/New Jersey/1976 H1N1 swine influenza vaccine neutralized 2009 H1N1. Forty five percent also neutralized A/New Caledonia/20/1999 H1N1, a strain used in seasonal influenza vaccines during the 2000/01–2006/07 seasons. Among adults aged 48–64 who received the swine influenza vaccine in 1976 and recent seasonal influenza vaccines during the 2004/05–2008/09 seasons, 83% had sera that neutralized 2009 H1N1. However, 68% of age-matched subjects who received the same seasonal influenza vaccines, but did not receive the 1976 swine influenza vaccine, also had sera that neutralized 2009 H1N1. Sera from both 1976 and contemporary cohorts frequently had cross-neutralizing antibodies to 2009 H1N1 and A/New Caledonia/20/1999 that mapped to hemagglutinin subunit 2 (HA2). A conservative mutation in HA2 corresponding to a residue in the A/Solomon Islands/3/2006 and A/Brisbane/59/2007 H1N1 strains that circulated in the 2006/07 and 2007/08 influenza seasons, respectively, abrogated this neutralization. These findings highlight a cross-neutralization determinant influenced by a point mutation in HA2 and suggest that HA2 may be evolving under direct or indirect immune pressure.

Antibodies to the A27 Protein of Vaccinia Virus Neutralize and Protect against Infection but Represent a Minor Component of Dryvax Vaccine-Induced Immunity

The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

Characterization of lentiviral pseudotypes with influenza H5N1 hemagglutinin and their performance in neutralization assays

Pseudotype reporter viruses are being used as safe, quantitative, and high-throughput tools for assessing antibody neutralization for many viruses, including influenza. However, characterization of pseudotypes containing influenza hemagglutinin (HA-pseudotypes) is needed before this system is widely adopted for evaluating neutralizing antibodies in sera following vaccination or infection. In this report HA-pseudotype stocks were analyzed for HA content, stability, and performance in neutralization assays under various conditions. HA-pseudotypes produced with HA genes of H5 strains representing clades 1, 2.2, and 2.3.4 consistently contain similar HA contents, and infectivity was not greatly affected by the purity of the HA-pseudotype preparations or variations in storage conditions. HA-pseudotype neutralization titers using a reference serum panel were also consistent across a wide range of dilutions of HA-pseudotype stocks and correlated well with results from microneutralization assays involving replicating influenza. Concentrated HA-pseudotypes were further shown to work well in hemagglutination inhibition assays. Finally, antisera elicited by genetically modified HA, with changes in the polybasic cleavage site that have been used in some H5 vaccines and reduce pathogenicity, gave identical neutralization titers against HA-pseudotypes with wild type or modified HA. These findings support continued development of HA-pseudotypes as a robust tool for analyzing sera in vaccine and serologic studies.