Frequency and Phenotype of Human Immunodeficiency Virus Envelope-Specific B Cells from Patients with Broadly Cross-Neutralizing Antibodies

LIR, NIAID, National Institutes of Health, Bldg. 10, Rm. 7N246, 10 Center Dr., Bethesda, MD 20892, USA.
Journal of Virology (Impact Factor: 4.44). 11/2008; 83(1):188-99. DOI: 10.1128/JVI.01583-08
Source: PubMed


Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27(hi) CD38(hi) plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27(+) and surface IgG(+). These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.

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    • "−/low CD27 hi CD38 hi cells (Wrammert et al., 2008; Doria-Rose et al., 2009). However, these markers did not translate into a useful approach for the analysis of macaque plasmablasts. "
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    ABSTRACT: Over 100 broadly neutralizing antibodies have been isolated from a minority of HIV infected patients, but the steps leading to the selection of plasma cells producing such antibodies remain incompletely understood, hampering the development of vaccines able to elicit them. Rhesus macaques have become a preferred animal model system used to study SIV/HIV, for the characterization and development of novel therapeutics and vaccines as well as to understand pathogenesis. However, most of our knowledge about the dynamics of antibody responses is limited to the analysis of serum antibodies or monoclonal antibodies generated from memory B cells. In a vaccine setting, relatively little is known about the early cellular responses that elicit long-lived plasma cells and memory B cells and the tools to dissect plasmablast responses are not available in macaques. In the current study, we show that the majority (> 80%) of the vaccine-induced plasmablast response are antigen-specific by functional ELISPOT assays. While plasmablasts are easily defined and isolated in humans, those same phenotypic markers have not been useful for identifying macaque plasmablasts. Here we describe an approach that allows for the isolation and single cell sorting of vaccine-induced plasmablasts. Finally, we show that isolated plasmablasts can be used to efficiently recover antigen-specific monoclonal antibodies through single cell expression cloning. This will allow detailed studies of the early plasmablast responses in rhesus macaques, enabling the characterization of both their repertoire breadth as well as the epitope specificity and functional qualities of the antibodies they produce, not only in the context of SIV/HIV vaccines but for many other pathogens/vaccines as well.
    Full-text · Article · Nov 2014 · Journal of Immunological Methods
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    • "Env glycans influence these interactions [31-35] and, thus, impact HIV-1 infectivity [36,37]. In this regard, the vaccine trial RV144 identified a potentially protective epitope of HIV-1 Env in the gp120 V1/2 loop [38,39] and V1/2 loop glycan (N160) that contributes to formation of a quarternary-structure epitope recognized by new class of broadly neutralizing monoclonal antibodies of PG family [10,40]. Although glycans influence protein folding, potentially affecting the conformation of surface-exposed epitopes involved in antibody binding, N-glycans also serve as epitopes or part of epitopes for some broadly neutralizing antibodies [40-54]. "
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    ABSTRACT: Background HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. Methods Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. Results We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. Conclusion Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen.
    Full-text · Article · Aug 2014 · AIDS Research and Therapy
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    • "Nevertheless, the role of non-neutralizing anti-env Abs in HIV-1 infection remains unclear. More than 95% of the body’s CD4+ T cells reside in lymphoid tissues, which are the major sites for HIV-1 replication, CD4+ T cell depletion [5], and development of anti-env Ab-secreting B cells [3], [6]. CD4+ T cells continuously travel between the blood, the lymphatic system, and lymph nodes (LNs) and re-circulate into the blood over a period of approximately 1 d [7]–[9]; therefore, most peripheral blood CD4+ T cells are recent emigrants from the LNs. "
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    ABSTRACT: Peripheral blood CD4(+) T cells in HIV-1(+) patients are coated with Ig. However, the causes and consequences of the presence of Ig(+) CD4(+) T cells remain unknown. Previous studies have demonstrated the rapid turnover of viral receptors (VRs) on lymphoma and tumor cells. The present study investigates the turnover of VRs on peripheral quiescent CD4(+) T cells (qCD4s), which are the most abundant peripheral blood CD4(+) T cells. Utilizing pharmacological and immunological approaches, we found that the turnover of VRs on qCD4s is extremely slow. As a result, exposure to gp120 or HIV-1 virions in vitro causes gp120 to remain on the surface for a long period of time. It requires approximately three days for cell-bound gp120 on the surface to be reduced by 50%. In the presence of patient serum, gp120 forms surface immune complexes (ICs) that are also retained for a long time. Indeed, when examining the percentages of Ig(+) CD4(+) T cells at different stages of HIV-1 infection, approximately 70% of peripheral resting CD4(+) T cells (rCD4s) were coated with surface VRs bound to slow-turnover gp120-Ig. The levels of circulating ICs in patient serum were insufficient to form surface ICs on qCD4s, suggesting that surface ICs on qCD4s require much higher concentrations of HIV-1 exposure such as might be found in lymph nodes. In the presence of macrophages, Ig(+) CD4(+) T cells generated in vitro or directly isolated from HIV-1(+) patients were ultimately phagocytosed. Similarly, the frequencies and percentages of Ig(+) rCD4s were significantly increased in an HIV-1(+) patient after splenectomy, indicating that Ig(+) rCD4s might be removed from circulation and that non-neutralizing anti-envelope antibodies could play a detrimental role in HIV-1 pathogenesis. These findings provide novel insights for vaccine development and a rationale for using Ig(+) rCD4 levels as an independent clinical marker.
    Full-text · Article · Feb 2014 · PLoS ONE
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