William J. Honnen

The V1/V2 Domain of gp120 Is a Global Regulator of the Sensitivity of Primary Human Immunodeficiency Virus Type 1 Isolates to Neutralization by Antibodies Commonly Induced upon Infection

ABSTRACT A major problem hampering the development of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) is the resistance of many primary viral isolates to antibody-mediated neutralization. To identify factors responsible for this resistance, determinants of the large differences in neutralization sensitivities of HIV-1 pseudotyped with Env proteins derived from two prototypic clade B primary isolates were mapped. SF162 Env pseudotypes were neutralized very potently by a panel of sera from HIV-infected individuals, while JR-FL Env pseudotypes were neutralized by only a small fraction of these sera. This differential sensitivity to neutralization was also observed for a number of monoclonal antibodies (MAbs) directed against sites in the V2, V3, and CD4 binding domains, despite often similar binding affinities of these MAbs towards the two soluble rgp120s. The neutralization phenotypes were switched for chimeric Envs in which the V1/V2 domains of these two sequences were exchanged, indicating that the V1/V2 region regulated the overall neutralization sensitivity of these Envs. These results suggested that the inherent neutralization resistance of JR-FL, and presumably of related primary isolates, is to a great extent mediated by gp120 V1/V2 domain structure rather than by sequence variations at the target sites. Three MAbs (immunoglobulin G-b12, 2G12, and 2F5) previously reported to possess broad neutralizing activity for primary HIV-1 isolates neutralized JR-FL virus at least as well as SF162 virus and were not significantly affected by the V1/V2 domain exchanges. The rare antibodies capable of neutralizing a broad range of primary isolates thus appeared to be targeted to exceptional epitopes that are not sensitive to V1/V2 domain regulation of neutralization sensitivity.

Human Monoclonal Antibodies Specific for Conformation-Sensitive Epitopes of V3 Neutralize Human Immunodeficiency Virus Type 1 Primary Isolates from Various Clades

ABSTRACT The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3JR-CSF sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.

A human monoclonal antibody against the CD4-binding site of HIV1 gp120 exhibits potent, broadly neutralizing activity

A human monoclonal antibody (HuMAb) against HIV1, 1125H, was isolated from an asymptomatic, seropositive haemophiliac. This antibody was specific for gp120, and its binding to gp120 was inhibited by soluble CD4, indicating that its epitope was in or near the CD4-binding site. 1125H antibody recognized a variety of divergent HIV1 strains, including most laboratory strains tested as well as some early passage isolates. Commensurate with its specificity and high apparent affinity, 1125H exhibited potent neutralizing activity against IIIB, MN, RF and SF-2 strains. The epitope recognized by 1125H was destroyed by reduction of disulphide bonds, but not by removal of N-linked sugars. Thus, the epitope was conformationally determined and did not involve carbohydrate. Data from radioimmunoprecipitation/SDS-PAGE analysis of proteolytically cleaved viral lysate further indicated that the epitope of 1125H was not affected by cleavage at the V3 loop of gp120, provided that gp120 disulphide bonds remained intact. The potential use of HuMAb 1125H in passive immunotherapy against HIV is discussed as well as the importance of including its epitope in an AIDS vaccine.

Antibodies That Are Cross-Reactive for Human Immunodeficiency Virus Type 1 Clade A and Clade B V3 Domains Are Common in Patient Sera from Cameroon, but Their Neutralization Activity Is Usually Restricted by Epitope Masking

ABSTRACT Sera from human immunodeficiency virus type 1 (HIV-1)-infected North American patients recognized a fusion protein expressing a V3 loop from a clade B primary isolate virus (JR-CSF) but not from a clade A primary isolate virus (92UG037.8), while most sera from Cameroonian patients recognized both fusion proteins. Competition studies of consensus V3 peptides demonstrated that the majority of the cross-reactive Cameroonian sera contained cross-reactive antibodies that reacted strongly with both V3 sequences. V3-specific antibodies purified from all six cross-reactive sera examined had potent neutralizing activity for virus pseudotyped with envelope proteins (Env) from SF162, a neutralization-sensitive clade B primary isolate. For four of these samples, neutralization of SF162 pseudotypes was blocked by both the clade A and clade B V3 fusion proteins, indicating that this activity was mediated by cross-reactive antibodies. In contrast, the V3-reactive antibodies from only one of these six sera had significant neutralizing activity against viruses pseudotyped with Envs from typically resistant clade B (JR-FL) or clade A (92UG037.8) primary isolates. However, the V3-reactive antibodies from these cross-reactive Cameroonian sera did neutralize virus pseudotyped with chimeric Envs containing the 92UG037.8 or JR-FL V3 sequence in Env backbones that did not express V1/V2 domain masking of V3 epitopes. These data indicated that Cameroonian sera frequently contain cross-clade reactive V3-directed antibodies and indicated that the typical inability of such antibodies to neutralize typical, resistant primary isolate Env pseudotypes was primarily due to indirect masking effects rather than to the absence of the target epitopes.

Potent neutralization of primary HIV-1 isolates by antibodies directed against epitopes present in the V1/V2 domain of HIV-1 gp120.

Although it is known that some human immune sera possess potent neutralizing activities for primary viruses, the identity of the target epitopes mediating this neutralization is unknown, and currently available immunogens have not been able to induce such activities. Using recombinant fusion glycoproteins expressing native V1/V2 domains of gp120 we have found that sera from a subset of HIV-1-infected humans contain antibodies that recognize broadly conserved V1/V2 epitopes. Such antibodies were isolated from one human serum by affinity chromatography on a column containing a V1/V2 fusion protein, and shown to efficiently neutralize several macrophage-tropic HIV-1 isolates. Rodents immunized with the purified V1/V2 fusion protein produced antibodies reactive with unrelated V1/V2 fusion proteins and with heterologous gp120s. V1/V2-specific immunoglobulins isolated from sera of these animals by affinity chromatography also possessed potent neutralization activity for several primary HIV-1 isolates. These results indicate that the V1/V2 domain of HIV-1 gp120 contains conserved epitopes that mediate potent neutralization of primary viruses, and suggest that subunit vaccines that efficiently induce such antibodies may provide protective humoral immunity against clinically relevant HIV-1 isolates.

V3-Specific Polyclonal Antibodies Affinity Purified from Sera of Infected Humans Effectively Neutralize Primary Isolates of Human Immunodeficiency Virus Type 1

Although many human sera possess potent neutralizing activities for primary HIV-1 viruses, such activities are not efficiently induced by the current generation of vaccine candidates, and the epitopes mediating this neutralization are not known. The V3 loop of gp120 is believed to be the principal neutralization domain of laboratory-adapted viruses, but the importance of this region in neutralization of primary isolates is unclear. This question was explored using polyclonal anti-V3 antibodies purified by immunoaffinity methods from sera of HIV-1-infected patients. To include antibodies that might be directed against conformational and/or glycan-dependent epitopes not presented by synthetic peptides, the antibody isolations were performed with a fusion glycoprotein expressing the native V3 region of JR-CSF, a primary R5 isolate. V3-reactive antibody fractions from all eight sera examined showed potent neutralization of at least one of the three primary HIV-1 isolates tested; four of these antibody preparations neutralized all three primary viruses. For a number of serum-virus combinations 90% neutralization doses (ND(90)) between 1 and 5 microg/ml were obtained, and the most potent anti-V3 fraction had ND(50) values at or below 0.3 microg/ml for all three primary isolates. These neutralization activities against primary viruses were higher than those of potent monoclonal antibodies assayed in the same experiment. These data indicate that the V3 region can be an important neutralization target in primary isolates, and suggest that effective presentation of V3 epitopes in a vaccine formulation might induce protective humoral responses against natural infection by HIV-1.

Efficient Isolation of Novel Human Monoclonal Antibodies with Neutralizing Activity Against HIV-1 from Transgenic Mice Expressing Human Ig Loci

Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIVSF162 resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIVSF162 strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.

The C108g Epitope in the V2 Domain of gp120 Functions as a Potent Neutralization Target When Introduced into Envelope Proteins Derived from Human Immunodeficiency Virus Type 1 Primary Isolates

ABSTRACT Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.

Biochemical characterization of cell-associated and extracellular products of the Friend spleen focus-forming virus env gene.

The mature product of the env gene of Friend spleen focus-forming viruses (F-SFFV) is efficiently released from both leukemia cells and infected fibroblasts. Analyses of the kinetics of env protein synthesis and secretion in NRK cells infected with the Lilly-Steeves strain of SFFVp indicated that this product, gp65, was formed rapidly and remained stably associated with cells for up to 4 hr, at which point it was first detected in supernatant medium. By 12 hr after synthesis, greater than 95% of gp65 was found extracellularly. The release of this component was effectively blocked by 10 mM 1-deoxynojirimycin, an inhibitor of oligosaccharide processing, demonstrating a requirement for processing of high mannose precursor oligosaccharides in the secretion of gp65. Similar oligosaccharide substituents were found on cell-associated and extracellular forms of gp65. Enzymatic deglycosylation experiments demonstrated that in addition to the predicted four N-linked oligosaccharides, gp65 contains O-linked carbohydrates which are resistant to the action of peptide N-Glycanase F, but sensitive to neuraminidase and O-Glycanase. These structures may be related to O-linked oligosaccharides previously found on the env gene products of murine leukemia viruses. Comparison of the sizes of the deglycosylated forms of cell-associated and supernatant gp65 demonstrated that the extracellular molecules are approximately 3 kDa smaller than the cell-associated components. These data suggest the involvement of proteolysis at a C-terminal site in the release of gp65 from the plasma membrane.

A sensitive radioimmunoprecipitation assay for human immunodeficiency virus (HIV)

A sensitive and efficient radioimmunoprecipitation procedure is described which provides an alternative to Western blotting assays for characterizing antibodies directed against human immunodeficiency viruses (HIV-1). Reaction of solubilized preparations of HTLV-III with 125I-labeled Bolton-Hunter reagent leads to the efficient labeling of all of the major virus-specific proteins, including gp120, gp41, RT (p66/p51), p24, and p17. These labeled proteins are readily immunoprecipitated by immune human sera, by specific sera derived from hyperimmunized animals, and by monoclonal antibodies. This procedure, referred to as BH-RIP, provides a simple assay for characterizing and titering antibodies against HIV which is equivalent in specificity, and more sensitive and efficient than the Western blotting method. In addition, viral proteins labeled in this way are suitable for biochemical studies. In one such application, the number of high-mannose and complex oligosaccharide side chains of gp120 and gp41 were determined by examining the sensitivities of the two viral glycoproteins labeled by this procedure to the glycosidases Endo H and PNGase F.