HIV-1gp120: A Novel Viral B Cell Superantigen

The envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, has recently been characterized as a novel immunoglobulin superantigen (Ig-SAg) [1,2]. Analogous to the interaction of SAgs with T cells, gp120 binds to an unusually large proportion of immunoglobulins (Igs) from HIV-uninfected individuals; most, if not all of these Igs are members of the VH3 family [3]. Functionally, gp 120 preferentially stimulates VH3 B cells in vitro. This stimulation correlates with an in vivo VH3 activation during HIV infection. Curiously, this initial activation is followed by a subsequent depletion of VH3-expressing B cells as individuals progress to AIDS.

In this article we will review our current understanding of the superantigenic properties of HIV gp120. Specifically we will focus on structural aspects of the binding interaction, on the ontological development of these superantigen-binding antibodies, and on potential roles that this unconventional Ig-pathogen interaction might play in the pathogenesis of HIV-induced disease.     

A human anti-HIV autoantibody enhances EBV transformation and HIV infection

A highly specific, human IgG mAb, F223, which reacts with both HIV-1-infected cells and uninfected lymphoid cells, has been derived. F223 reacts with gp120 but fails to neutralize viral infection. The antibody does enhance HIV-1 infection in a complement-dependent manner. The autoantigen recognized by F223 is expressed on a small percentage of T cells and NK cells and the majority of B cells. Immunoprecipitation demonstrates F223 reactivity with an as of yet unidentified 159-kDa protein in uninfected lymphoid cells. This reactivity with uninfected cells is inhibited by free gp120 demonstrating the cross-reactive nature of this antibody. The F223 light chain demonstrates strong homology to VLlambda2 family genes whereas the heavy chain is most homologous (84%) to the germline gene VH3-H.11. In vivo usage of VH3 family genes by F223 and an anti-HIV-1 (gp41) human mAb, 3D6, with related autoreactivity, suggests that VH3 sequences may be important components of potentially pathogenic human anti-HIV-1 envelope autoantibodies. F223 was isolated from an HIV-1 infected individual with lymphoma and in vitro F223 significantly enhances EBV transformation of normal B cells and increases immunoglobulin production without affecting B cell proliferation. Characterization of this antibody response may provide important insights and mechanistic information on HIV pathogenesis.     

gp120- and TNF-alpha-induced modulation of human B cell function: proliferation, cyclic AMP generation, Ig production, and B-cell receptor expression

BACKGROUND: It is well known that HIV-1 infection induces profound alterations in the immune system, including hyperactivation of B cells. TNF-alpha induces HIV-1 replication and immunodysregulation, including polyclonal B-cell activation. OBJECTIVE: We sought to determine the effects of surface-binding HIV-1 envelope glycoprotein (gp120) and TNF-alpha on human B-cell function. METHODS: HIV-1 seronegative peripheral blood human B cells were purified and activated by CD40 mAb and IL-4. In vitro studies of B-cell proliferation, cyclic AMP (cAMP) generation, receptor expression, and Ig production were performed. RESULTS: gp120, an Ig superantigen, stimulated HIV-1 seronegative and HIV-1 seropositive human B-cell cAMP generation, proliferation, and Ig production. These gp120-induced B-cell responses were demonstrated to be specific as evidenced by the abrogation of the stimulatory response in the presence of anti-gp120 mAb, blocking of CD4 resulting in no change on gp120-induced B-cell responses, and the binding of gp120 in these B cells. TNF-alpha also stimulated cAMP generation, proliferation, and Ig production in B cells, and the binding of gp120 to these B cells stimulated by TNF-alpha further enhanced cell proliferation, cAMP generation, and Ig production. Antigenic expression of the B-cell receptor CD79b was down-regulated by gp120 but was not altered by the addition of TNF-alpha. CONCLUSION: gp120 modulation of TNF-alpha-induced B-cell receptor- and cAMP-mediated signal transduction events may be involved in the B-cell abnormalities observed in HIV-1 infection.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

B Cell Superantigens: Potential Modifiers of the Normal Human BCell Repertoire

Staphylococcal protein A (SPA), HIV gp120, and staphylococcal enterotoxins (SE) are B cell superantigens that induce V_H specific B cell responses. In addition, the red blood cell antigens, i/I, have some features of a B cell superantigen. Binding of SPA, SE and HIV gp120 are V_H family specific, whereas binding of i/I is V_H gene specific. SPA and HIV gp120 function by stimulating V_H3-expressing B cells, whereas SE appear to function by enhancing survival of the appropiate V_H-expressing B cells. Moreover, HIV gp120 has been shown to delete VH3-expressing B cells. In this review, we describe evidence that shows how these superantigens may play a role in shaping the normal B cell repertoire.     

Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.     

Molecular Determinants of the Human Antibody Response to HIV-1: Implications for Disease Control

Various aspects of the immune response to HIV-1 infection remain unclear. While seropositive subjects generally mount a strong humoral response, the antibodies produced are not effective in halting disease progression. Molecular characterization of the antibody repertoire specific for HIV-1 antigens represents an approach to further our understanding of the mechanisms involved in mounting a humoral immunity in this infection. Recently, the content, structure, and organization of the human immunoglobulin-variable gene loci have been elucidated and a number of laboratories have characterized the variable gene elements of human anti-HIV-1 antibodies derived from infected persons by cell fusion or by Epstein–Barr virus transformation. The results show evidence for extensive somatic mutations that lead to preferential amino acid substitutions in the hypervariable regions, an indication of an antigen-driven process. Multiple other molecular events also are engaged in generating antibody diversity, including various types of fusions of variable genes, usage of inverted diversity genes, and addition of extragenomic nucleotides. Most importantly, there is a paucity of antibodies expressing the major V_H3 gene family, which could result from the capacity of gp120 to act as superantigen for human B cells. This V_H3⁺ antibody deficit also has been observed in B cells isolated ex vivo from the patients. Since V_H3⁺ antibodies play an essential role in immune defense against infections, the abnormalities observed in HIV-1 infection may predispose to opportunistic infections and further compromise the immune defense mechanisms of the subjects.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4⁺ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4⁺ T cell attrition in vivo.     

Molecular analysis of human immunoglobulin heavy chain variable region associated determinants recognized by anti-VH3 antibodies 7B4, B6 and D12

7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3-30, V3-30.3 and V3-33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3-11 and V3-7 genes; D12 also reacts with some V3-43 gene Ig. We show here, by site-directed mutagensis, that the lysine at position 57 in the complementarity-determining region 2 (CDR-2) of the V3-30 gene product is crucial for epitope recognition by all three anti-VH3 MoAbs. Further analysis of the amino-acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR-2. Comparing the efficiency of 7B4 reactivity with VH3 gene-encoded human Ig indicates that amino-acid position 4 in the frame region 1 (FR-1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3-30, V3-30.3 and V3-33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)-2, SIV and HTLV-4. We also report that other tetrapeptide sequences found on the 56-59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV-1 isolates. The possible significance of these observations is discussed.     

The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.     

Utility of various commercially available human immunodeficiency virus (HIV) antibody diagnostic kits for use in conjunction with efficacy trials of HIV-1 vaccines.

There is a need for human immunodeficiency virus (HIV) screening assays which will distinguish uninfected HIV vaccine recipients from HIV-infected individuals. Commercial screening kits were used to test serum samples from low- and high-risk participants in clinical trials before and after immunization with various recombinant HIV type 1 (HIV-1) envelope glycoprotein 120 (gp120) candidate vaccines. All kits were 100% sensitive in detecting HIV infection. Both Murex Single Use Diagnostic System and United Biomedical, Inc., HIV type 1 or 2 (HIV-1/2) enzyme immunoassay (EIA) kits, which detect antibodies to HIV-1 gp41, were 98 to 100% specific when used to screen baseline or recombinant gp120-vaccinated populations as vaccine-induced antibodies to gp120 were nonreactive in these tests. The Abbott HIVAB HIV-1 EIA (lysate of whole infected cells, reactive with anti-gp120 antibodies) gave high levels of reactivity due to vaccine-induced antibodies and a high baseline rate of false positives (12 of 83) among nonvaccinated high-risk volunteers. Assays containing only gp41 and p24 solid-phase components are compatible with gp120-based vaccines but are unlikely to be useful in a similar role for vaccines containing gp160, gp41, or gp120 plus p24 antigens. Efficacy trials must be designed in concert with available diagnostic screening assays to avoid problems caused by vaccine-induced seroconversion in high-risk populations.     

Natural Human Antibodies Retrieved by Phage Display Libraries from Healthy Donors: Polyreactivity and Recognition of Human Immunodeficiency Virus Type 1 gp120 Epitopes

We have characterized the human natural antibody repertoire that contains antibodies recognizing the human immunodeficiency virus type 1 (HIV-1) gp120. A panel of monovalent antigen-binding fragments (Fab) selected from IgM and IgG isotype libraries generated from peripheral blood mononuclear cells (PBMC) of a healthy, HIV-1 noninfected individual was analysed, reflecting that only IgM, but not IgG, Fab were able to recognize HIV-1 gp120. The IgM Fab antibodies were not restricted to any particular heavy chain variable region (VH) germ line gene. However, the recognition of gp120 is associated to polyreactive antibodies and all display low-affinity interaction. This correlates with the absence of any maturation process as somatic mutation or isotype switch as the nucleotide sequence analysis of the variable regions reveals they are expressed near to germline configuration. In addition, none of the antibodies showed any neutralizing activity on HIV-1-infected lymphocytes, reflecting that the natural anti-gp120 repertoire is not sufficient to neutralize HIV infection.     

Human monoclonal and polyclonal anti-human immunodeficiency virus-1 antibodies share a common clonotypic specificity.

Human monoclonal and purified polyclonal anti-human immunodeficiency virus (HIV)-1 antibodies were tested for binding to a murine monoclonal anti-idiotypic antibody (1F7, IgM, kappa). Four human monoclonal anti-p24 and three human monoclonal anti-gp120 antibodies express the 1F7 clonotype, while one human monoclonal anti-gp41 antibody does not bind to 1F7. Affinity-purified anti-p24 and anti-gp120 antibodies from HIV-1-infected individuals also react with 1F7. Western blot analysis and enzyme-linked immunosorbent assay confirmed that 1F7 reacts with human antibodies of different HIV-1 antigen specificities. A survey of sera from 329 HIV-1-infected individuals showed binding to 1F7 in 239 sera (72.6%) while 1F7 was not reacting with 109 HIV-1-negative sera. These results show that 1F7 idiotype is an HIV-1 infection-associated clonotypic marker shared by anti-HIV-1 antibodies with different epitope specificites.     

HIV-1 gp41 envelope IgA is frequently elicited after transmission but has an initial short response half-life

Prevention of HIV-1 transmission at mucosal surfaces will likely require durable pre-existing mucosal anti-HIV-1 antibodies (Abs). Defining the ontogeny, specificities and potentially protective nature of the initial mucosal virus-specific B-cell response will be critical for understanding how to induce protective Ab responses by vaccination. Genital fluids from patients within the earliest stages of acute HIV-1 infection (Fiebig I–VI) were examined for multiple anti-HIV specificities. Gp41 (but not gp120) Env immunoglobulin (Ig)A Abs were frequently elicited in both plasma and mucosal fluids within the first weeks of transmission. However, shortly after induction, these initial mucosal gp41 Env IgA Abs rapidly declined with a t_1/2 of ∼2.7 days. B-cell-activating factor belonging to the TNF family (BAFF) was elevated immediately preceding the appearance of gp41 Abs, likely contributing to an initial T-independent Ab response. HIV-1 transmission frequently elicits mucosal HIV-1 envelope-specific IgA responses targeted to gp41 that have a short half-life.     

Molecular characterization of the circulating anti-HIV-1 gp120-specific B cell repertoire using antibody phage display libraries generated from pre-selected HIV-1 gp120 binding PBLs

Human bone marrow is a major repository for maturated antibody-secreting plasma cells, which produce the majority of the antibodies found in serum, making it an attractive source for generating human immune antibody libraries. Unfortunately, bone marrow is not always readily available and, although human immune libraries can be generated from circulating B cells, the low frequency of antigen-specific B cells in the circulation yield few monoclonal antibodies of interest. We used a pre-selection strategy to enrich for antigen-specific B cells prior to library generation, and applied this approach to evaluate, at a molecular level, the nature of the human anti-HIV-1 gp120 repertoire encoded by circulating B cells. IgG antibody phage display libraries were generated from HIV-1 seropositive individuals using either affinity-selected anti-gp120 IgG-bearing circulating B cells, predominantly exhibiting memory/activated B cell phenotype, or unselected PBMCs. These libraries were selected against HIV-1 gp120, resulting in isolation of a panel of gp120-specific antibodies. Whereas only 2 gp120-specific antibodies were retrieved from the non-pre-selected HIV-1 library, 9 gp120-specific antibodies were retrieved from the 10-fold smaller library generated from the pre-selected B cells, demonstrating the feasibility of the approach. The anti-gp120 antibodies derived from the circulating B cells of HIV-1 donors generally resembles those from bone marrow plasma cells with respect to epitope specificity, affinity and neutralization ability. They exhibit high affinity for gp120, are directed against a variety of epitopes, but rarely exhibit the ability to neutralize HIV-1.     

Insertion of a short human immunodeficiency virus (HIV)-2 gp36 sequence into an HIV-1 p24 recombinant protein results in a polypeptide with potent and TCRBV-restricted T cell triggering activity

In the present work we investigate whether artificial alterations of the structure of an inactive retrovirus-encoded protein could transform it in a superantigen. As a model system we used a recombinant human immunodeficiency virus (HIV)-1 p24 protein and two of its variants in which a short peptide corresponding to sequences of gp41 of HIV-1 (HIV-1 p24*) or gp36 of HIV-2 (HIV-1-2 p24*) has been inserted nearby the carboxy-terminal end of HIV-1 p24. As expected both HIV-1 p24 and HIV-1 p24* were inactive, while HIV-1-2 p24* was a potent inducer of human, but not murine, T cell proliferation. The possibility that the observed activity was due to contaminants was ruled out since the proliferative response could be specifically inhibited by a monoclonal anti-p24 antibody and by a peptide encompassing the area of HIV-1 p24/HIV-2 gp36 junction. Furthermore, the data exclude the possibility that the gp36 insertion is per se responsible for the observed proliferative activity. The analysis of the functional, phenotypic and molecular properties of the responding cells demonstrated that the response was class II dependent and that the activated cells were predominantly CD4⁺CD8⁻ expressing a strongly biased repertoire of TCRBV segments. Collectively, these data strongly suggest that the HIV-1-2 p24* fusion protein shares common functional properties typical of superantigen molecules. Thus, our demonstration that a viral protein can be transformed into a superantigen simply by the insertion of a short peptide at the carboxy-terminal end has important implications for understanding the mode of action of retrovirus-encoded superantigens.     

Fine specificity of IgG subclass response to group antigens in HIV-1-infected patients

There is an association between the clinical stage of HIV-1 infection and the presence of antibodies against viral gag proteins (p17 and p24). The IgG subclass (G1 and G3) pattern against these antigens was analysed in stable patients and HIV patients progressing to AIDS. Antibodies were analysed with whole viral or peptide ELISA (using sequentially overlapping peptides) and Western Blots. IgG1 was found to be the dominant anti-HIV-1 IgG subclass and IgG1 antibodies declined in progressing patients against all HIV antigens evaluated in Western blot, including p17, p24, p31, gp41, p64, gp120 and gp160. In contrast IgG3 antibodies, which were found to be predominantly directed against gag proteins, and which could be detected in almost all patients, remained in the circulation during disease progression. By peptide assays distinct immunogenic regions were found in p17 in contrast to more evenly distributed epitopes in p24. A decreased divergence of antibody reactivity to both p17 and p24 peptides in the group of patients who developed AIDS was seen. No reaction to any single gag epitope related to disease progression. The difference between IgG1 and IgG3 anti-gag antibodies in relation to clearance during disease progression may depend on different properties of immune complexes formed by these two IgG subclasses.