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.

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.

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.

Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults.

BACKGROUND: The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). METHODOLOGY/PRINCIPAL FINDINGS: A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. CONCLUSIONS/SIGNIFICANCE: The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. TRIAL REGISTRATION: ClinicalTrials.gov NCT00057525.

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1.

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC(50)s ranging from 0.1 to 7.4 µg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions.

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

PURPOSE OF REVIEW: In 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 FINDINGS: The 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. SUMMARY: HIV-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.

Human erythrocytes selectively bind and enrich infectious HIV-1 virions.

Although CD4(+) cells represent the major target for HIV infection in blood, claims of complement-independent binding of HIV-1 to erythrocytes and the possible role of Duffy blood group antigen, have generated controversy. To examine the question of binding to erythrocytes, HIV-1 was incubated in vitro with erythrocytes from 30 healthy leukapheresis donors, and binding was determined by p24 analysis and adsorption of HIV-1 with reduction of infectivity for CD4(+) target cells. All of the cells, regardless of blood group type, bound HIV-1 p24. A typical preparation of erythrocytes bound <2.4% of the added p24, but erythrocytes selectively removed essentially all of the viral infectivity as determined by decreased infection of CD4(+) target cells; however, cell-associated HIV-1 was approximately 100-fold more efficient, via trans infection, than unadsorbed virus for infection of CD4(+) cells. All of the bound HIV-1 p24 was released by treatment of the cells with EDTA, and binding was optimized by adding Ca(2+) and Mg(2+) during the washing of erythrocytes containing bound HIV-1. Although the small number of contaminating leukocytes in the erythrocyte preparation also bound HIV-1 p24, there was no significant binding to CD4, and it thus appears that the binding occurred on leukocytes at non-CD4 sites. Furthermore, binding occurred to erythrocyte ghosts from which contaminating leukocytes had been previously removed. The results demonstrate that erythrocytes incubated in vitro with HIV-1 differentially adsorb all of the infectious HIV-1 virions (as opposed to non-infectious or degraded virions) in the absence of complement and independent of blood group, and binding is dependent on divalent cations. By analogy with HIV-1 bound to DC-SIGN on dendritic cells, erythrocyte-bound HIV-1 might comprise an important surface reservoir for trans infection of permissive cells.

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.

Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination.

In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a "gatekeeper" for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved.

Monoclonal antibodies to phosphatidylinositol phosphate neutralize human immunodeficiency virus type 1: role of phosphate-binding subsites.

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.

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.

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.

Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha.

Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Igalpha. We postulated that the Igalpha nonimmunoreceptor tyrosine-based activation motif tyrosines, Y(176) and Y(204), contributed to receptor trafficking. Igalpha(YDeltaF(176,204))/Igbeta receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y(176)/Y(204) recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Igalpha(YDeltaF(176,204))/Igbeta with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.

Lipid rafts unite signaling cascades with clathrin to regulate BCR internalization.

A major function of the B cell is the internalization of antigen through the BCR for processing and presentation to T cells. While there is evidence suggesting that lipid raft signaling may regulate internalization, the molecular machinery coordinating these two processes remains to be defined. Here we present a link between the B cell signaling and internalization machinery and show that Src-family kinase activity is required for inducible clathrin heavy chain phosphorylation, BCR colocalization with clathrin, and regulated internalization. An analysis of different B cell lines shows that BCR uptake occurs only when clathrin is associated with rafts and is tyrosine phosphorylated following BCR crosslinking. We therefore propose that lipid rafts spatially organize signaling cascades with clathrin to regulate BCR internalization.