[Show abstract][Hide abstract]ABSTRACT: HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.
Preview · Article · Dec 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine. Furthermore, serological analyses do not provide information regarding how repeated Ag inoculation impacts the expansion and maintenance of Env-specific B cell subpopulations. Therefore, we developed a highly sensitive Env-specific B cell ELISPOT system, which allows the enumeration of Ab-secreting cells (ASC) from diverse anatomical compartments directed against different structural determinants of Env. In this study, we use this system to examine the evolution of B cell responses in mice immunized with engineered Env trimers in adjuvant. We demonstrate that the relative proportion of ASC specific for defined structural elements of Env is altered significantly by homologous booster immunizations. This results in the selective expansion of ASC directed against the variable regions of Env. We suggest that the B cell specificity and compartment analysis described in this study are important complements to serological mapping studies for the examination of B cell responses against subspecificities of a variety of immunogens.
Full-text · Article · Oct 2009 · The Journal of Immunology
[Show abstract][Hide abstract]ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.
[Show abstract][Hide abstract]ABSTRACT: Binding titers of sera from rabbits immunized with envelope variants as determined by ELISA. Affinity purified coreV3S protein (2 µg/ml) was coated on ELISA plates, reacted with fivefold serial dilutions of different immune sera and detected with anti-rabbit IgG-peroxidase conjugated secondary antibody. Arrows indicate end point titers, defined as the last reciprocal serum dilution at which the optical density signal was greater than twofold over the signal detected with the preimmune sera. A. Comparison of titers following two, three and four inoculations of coreV3S protein. B. Comparison of titers among different groups of immune rabbit sera following four inoculations.
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[Show abstract][Hide abstract]ABSTRACT: Neutralization profile of fivefold diluted rabbit immune sera tested against a panel of HIV-1 and HIV-2 isolates. All sera tested were collected after four inoculations. Neutralization by preimmune sera was used as negative control for serum reactivity.
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[Show abstract][Hide abstract]ABSTRACT: ELISA analysis comparing inhibition of sCD4 binding to envelope glycoprotein by various groups of rabbit immune sera (im.s.) collected after four inoculations. A. Validation of the sCD4-inhibition assay. ELISA plates coated with core gp120 (2 µg/ml) protein were preincubated with fivefold dilutions of rabbit immune sera or ligands, reacted with 0.8 µg/ml of sCD4 followed by biotinylated guinea pig IgG anti-CD4, and detected with HRP-conjugated streptavidin. BSA-immunized rabbit serum and WTgp120-immunized rabbit serum were used as negative and positive control respectively for serum interactions. Unlabeled IgGb12 and IgG17b were used as controls for ligand binding. Binding of sCD4 in presence of the lowest concentration of BSA-immunized serum was considered 100%. Margins of error from duplicate wells were negligible. A. Validation of the sCD4-inhibition assay. B. Range of residual sCD4 binding to coreV3S protein in the presence of the highest concentration (20-fold dilution) of various immune sera. Values obtained were normalized against 100% binding in the presence of 2500-fold diluted BSA-immunized sera. The horizontal lines indicate mean values with standard errors of mean (SEM) for each group of sera. C. Same as in B except blocking of CD4 binding by each group of sera was detected with the corresponding protein immunogen coated on ELISA plate.
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[Show abstract][Hide abstract]ABSTRACT: Reducing SDS-PAGE of unmodified and stabilized core glycoproteins. All glycoproteins shown except the original core were purified by 17b affinity chromatography. The core was purified by b12 affinity chromatography because it is poorly recognized by 17b. The 17b antibody selects for a hyperglycosylated form of the modified core variants, in part accounting for the slightly slower migration of the 17b-purified glycoproteins in the gel.
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[Show abstract][Hide abstract]ABSTRACT: Elicitation of CD4i antibodies in guinea pigs following immunization with stabilized core variants. A. Schematic representation of immunogens used. B. IC50 titers of HIV-2 neutralization by guinea pig sera collected after 4 inoculations.
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[Show abstract][Hide abstract]ABSTRACT: ELISA analysis comparing inhibition of b12 binding to coreV3S protein by various groups of rabbit immune sera (im.s.). ELISA plates, coated with 2 µg/ml of protein, were preincubated with fivefold dilution of rabbit immune sera or ligands for 45 min at RT, reacted with biotinylated b12 (0.056 µg/ml of final concentration; M. Roederer, Conjugation of monoclonal antibodies, August 2004; http://www.drmr.com/abcon) for 30 min at RT and detected with 1∶250 dilution of HRP-conjugated streptavidin. BSA-immunized rabbit serum was used as negative control and either full-length gp120 (WTgp120)-immunized rabbit serum (panel A; Dey et al., 2007) or coreV3S-immunized rabbit serum (animal ID# 4; Panel B) were used as positive controls for serum interactions. Unlabeled IgGb12 and IgG17b were used as positive and negative controls respectively for ligand binding. Binding of b12 in the presence of the lowest concentration (2500-fold dilution) of BSA-immunized serum was considered 100%. Margins of error from duplicate wells were negligible. A. Validation of the b12-inhibition assay. B. Inhibition of b12 binding to coreV3S protein by immune sera tested over a range of dilution. C. Relative b12 binding to coreV3S protein in the presence of the highest concentration (20-fold dilution) of various immune sera. The dotted horizontal line indicates 100% b12 binding in the presence of 2500-fold diluted BSA-immunized sera. The mean values of b12 binding with standard errors of mean (SEM) for each group of sera are shown. Seven sera from coreV3S group and nine sera from each of 2CC, 3CC and 4CC groups were tested.
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[Show abstract][Hide abstract]ABSTRACT: Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum. However, little is known about the specificity and activity of these antibodies. To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads. We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site. Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120.
[Show abstract][Hide abstract]ABSTRACT: During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.
Full-text · Article · Dec 2008 · Journal of Virology
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Nov 2008 · Journal of Virology
[Show abstract][Hide abstract]ABSTRACT: We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.
[Show abstract][Hide abstract]ABSTRACT: The ability to readily elicit broadly neutralizing antibodies to HIV-1 remains elusive. We and others have hypothesized that interaction of the viral envelope glycoprotein (Env, gp120-gp41) with its receptor molecules could enhance the exposure of conserved epitopes that may facilitate the elicitation of broadly neutralizing antibodies. The Env-CD4-coreceptor complexes mediate HIV-1 entry into cells and serve as a major target for inhibitors of this process. To begin to evaluate their potential also as vaccine immunogens we produced relatively large amounts of complexes of purified recombinant soluble truncated Env, gp140(89.6) or gp120(89.6), with CD4 and CCR5 or CXCR4. We found that gp140(gp120)-CD4-CCR5 complexes are stable and immunogenic in mice transgenic for human CD4 and CCR5. They elicited anti-gp120 and anti-gp140 antibodies that inhibited an heterologous primary HIV-1 isolate (JR-FL) with two- to threefold higher neutralizing activity than those elicited by gp120 and gp140. The antibodies elicited by the complexes competed better with the antibodies X5 and CG10 but not with b12 for binding to gp120 and gp120-CD4 complexes compared to those elicited with gp140(120) alone. These findings suggest that stable purified Env-CD4-CCR5(CXCR4) complexes can be produced in relatively large amount sufficient for their further characterization that may help in the development of novel vaccines candidates.