Bruce K. Brown

Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy.

Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines

ABSTRACT A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.

Cross-clade neutralization patterns among HIV-1 strains from the six major clades of the pandemic evaluated and compared in two different models

A panel of paired primary virus isolates and envelope pseudoviruses from sixty strains representing six HIV-1 clades was tested for neutralization using pooled, clade-specific plasma in two prominently utilized neutralization platforms: a primary isolate assay using peripheral blood mononuclear cells (PBMC) and a pseudovirus assay using a reporter epithelial cell line. Using the PMBC assay, pairing of the antibody pool against homologous clade viruses generated the highest geometric mean neutralizing antibody titer in 4 out of 6 clades tested, and neutralization patterns showed numerous examples of reciprocal cross-recognition between antibody and viruses of specific clade pairs. In the pseudovirus assay, cross-clade neutralization was more limited, with fewer distinct cross-clade relationships evident. The clade C antibody pool was broadly cross-reactive, neutralizing the greatest number of viruses in both assays. These data highlight the importance of the neutralization assay format employed and suggest that clade C envelopes merit further evaluation for the elicitation of broadly neutralizing antibodies.

Monoclonal Antibodies to Phosphatidylinositol Phosphate Neutralize Human Immunodeficiency Virus Type 1: Role of Phosphate-Binding Subsites

ABSTRACT Both a murine monoclonal antibody to phosphatidylinositol phosphate (PIP) and a human monoclonal antibody (4E10) that is known to have broadly neutralizing capabilities against primary isolates of human immunodeficiency virus type 1 (HIV-1) bound to PIP, as determined by enzyme-linked immunosorbent assay. Each of the antibodies had antigen subsite binding specificities in aqueous medium for small phosphate-containing molecules and for inositol. The anti-PIP monoclonal antibody inhibited infection by two HIV-1 primary isolates in neutralization assays employing primary human peripheral blood mononuclear cells. The data suggest that PIP or related lipids having free phosphates could serve as targets for the neutralization of HIV-1.

The Role of Natural Killer (NK) Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses.

Impact of host cell variation on the neutralization of HIV-1 in vitro.

Purpose of reviewIn this review we present current advances in our understanding of HIV-1 neutralization assays that employ primary cell types, as compared with those that utilize cell lines and the newer, more standardized pseudovirus assays. A commentary on the challenges of standardizing in-vitro neutralization assays using primary cells is included. Recent findingsThe data from reporter cell line neutralization assays may agree with results observed in primary cells; however, exceptions have recently been reported. Multiple variables exist in primary cell assays using peripheral blood mononuclear cells from HIV-seronegative donors; in-vitro neutralization titers can vary significantly based on the donor cells used for assay targets and for virus propagation. Thus, more research is required to achieve validated primary cell neutralization assays. SummaryHIV-vaccine-induced antibody performance in the current neutralization assays may function as a ‘gatekeeper’ for HIV-1 subunit vaccine advancement. Development of standardized platforms for reproducible measurement of in-vitro neutralization is therefore a high priority. Given the considerable variation in results obtained from some widely applied HIV neutralization platforms, parallel evaluation of new antibodies using different host cells for assay targets, as well as virus propagation, is recommended until immune correlates of protection are identified.

Infection of human peripheral blood mononuclear cells by erythrocyte-bound HIV-1: Effects of antibodies and complement

Two human monoclonal antibodies, 4E10 and b12, were examined for antibody-dependent neutralization, or antibody-dependent complement (C)-mediated neutralization, of infection of peripheral blood mononuclear cells (PBMC) by either free HIV-1 or trans infection by HIV bound to erythrocytes. Neutralization of free HIV-1 by b12 was stronger than by 4E10, but b12 neutralized erythrocyte-bound HIV-1 less efficiently than cell-free virus. 4E10 did not neutralize erythrocyte-bound HIV-1 and at a low concentration it caused enhancement of infection. Antibody (4E10)-dependent C activation inhibited trans infection by erythrocyte-bound HIV-1, but caused enhanced infection with cell-free HIV-1 in the presence of erythrocytes. No effects of C were observed with b12. C-dependent enhancement in the presence of erythrocytes is proposed as due to binding of C3b-4E10-cell-free-HIV or C3d-4E10-cell-free-HIV to C receptor type 1 (CR1) on erythrocytes, or C receptor type 2 (CR2) on B cells in the PBMC, followed by trans infection of susceptible cells.

B Cell Depletion in HIV-1 Subtype A Infected Ugandan Adults: Relationship to CD4 T Cell Count, Viral Load and Humoral Immune Responses

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection.

Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling

Cultured primary peripheral blood mononuclear cells (PBMC) represent a potentially physiologic in vitro model of HIV-1 infection, but assessment of antibody-mediated HIV-1 neutralization using PBMC has been hindered by donor variability and lack of a sustainable individual PBMC source. To advance this model for HIV vaccine evaluation, intra- and inter-assay variability were assessed using monoclonal and polyclonal antibodies and PBMC targets from multiple HIV-seronegative donors. Inter-assay variability was introduced by using different PBMC for virus propagation, and more substantially, for assay targets. Neutralization titers varied by as much as 4 logs when using different individual donor PBMC as targets; variability was antibody-specific, with the greatest variation observed using an individual polyclonal plasma. Pooling of multiple PBMC donors significantly reduced median inter-assay variation to the level of intra-assay variation, suggesting a pathway forward for establishing a uniform, sustainable and standardized approach to the assessment of antibody function using a PBMC model.

P04-12. Characterization of leukopak PBMC phenotypes and biotypes for optimal performance in HIV-1 neutralization assays

Methods Leukopaks (N = 51) were analyzed using multi-parametric flow cytometry (LSRII cytometer, Becton Dickinson) to quantitate immune cell populations and surface markers. Viral titrations were performed using infectious molecular clones (IMC) (293T cell-derived or PBMC-passaged) and IC50 values were calculated using the Spearman-Karber formula. Productive replication in leukopaks was analyzed by p24 or luciferase expression; phenotypic correlates of viral permissiveness were assessed by Spearman correlation analyses.