Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6

Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1(89.6); MAbs giving significant neutralization at 2 to 10 microg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120(CD4bd)), 447-52D (anti-gp120(V3)), 2G12 (anti-gp120), and 670-D (anti-gp120(C5)). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1(89.6). Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120(V3) MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1(89.6); many anti-HIV-1 Abs act additively to mediate this biological function.     

Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site.

Synergistic neutralization of human immunodeficiency virus type 1 (HIV-1) was observed in studies using a chimpanzee anti-V2 monoclonal antibody (MAb), C108G, in combination with anti-V3 loop and anti-CD4 binding-site (bs) MAbs of different epitope specificities. C108G paired with either of two anti-V3 loop MAbs or either of two anti-CD4 bs MAbs synergistically neutralized both the uncloned IIIB and clonal HXB2 strains of virus in H9 target cells. Synergism was quantitated by calculation of combination indices. Significant synergy with a given MAb pair was seen over a range of MAb ratios, with the optimal effect centering around the ratio at which the MAbs were equipotent for a given HIV-1 strain (on the basis of the 50% neutralization titer). In preliminary experiments with monocytotropic strains of HIV-1 in peripheral blood mononuclear cell targets, significant synergism was also observed between anti-V2-anti-V3 and anti-V2-anti-CD4 bs MAb pairs. Synergism by all MAb pairs tested was greater against heterogeneous isolates of HIV-1 (IIIB and Ba-L) than against clonal isolates (HXB2 and NLHXADA), suggesting that strain broadening may be a component of the synergism observed against the heterogeneous isolates. In addition, conformational changes in gp120 upon binding of one or both MAbs may result in increased affinity or exposure of the epitope of one or both MAbs. Finally, a three-MAb combination of C108G, an anti-V3 MAb, and an anti-CD4 bs MAb was more effective in neutralizing the HXB2 strain of HIV-1 than any of the three two-MAb combinations within this trio, as determined by the dose reduction indices of each MAb required to achieve a given level of neutralization. This is the first report of synergistic neutralization of HIV-1 by a three-MAb combination composed of MAbs directed against the three major neutralization epitope clusters in gp120. Implications for vaccine design and for immunoprophylaxis and immunotherapy with a combination of MAbs are discussed.     

Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.     

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought.     

Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies

The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4(+)-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4(+)-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4(+) T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease.     

Synergistic Neutralization of Simian-Human Immunodeficiency Virus SHIV-vpu+ by Triple and Quadruple Combinations of Human Monoclonal Antibodies and High-Titer Anti-Human Immunodeficiency Virus Type 1 Immunoglobulins

We have tested triple and quadruple combinations of human monoclonal antibodies (MAbs), which are directed against various epitopes on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, and a high-titer anti-HIV-1 human immunoglobulin (HIVIG) preparation for their abilities to neutralize a chimeric simian-human immunodeficiency virus (SHIV-vpu⁺). This virus encodes the HIV-1 strain IIIB env, tat, rev, and vpu genes. The quantitative nature of the Chou-Talalay method (Adv. Enzyme Regul. 22:27–55, 1984) allows ranking of various combinations under identical experimental conditions. Of all triple combinations tested, the most potent neutralization was seen with MAbs 694/98D plus 2F5 plus 2G12 (directed against domains on V3, gp41, and gp120, respectively) as measured by the total MAb concentration required to reach 90% neutralization (90% effective concentration [EC₉₀], 2.0 μg/ml). All triple combinations involving MAbs and/or HIVIG that were tested yielded synergy with combination index values of <1; the dose reduction indices (DRIs) ranged from 3.1 to 26.2 at 90% neutralization. When four MAbs (the previous three plus MAb F105, directed against the CD4 binding site) were combined, higher neutralization potency (EC₉₀, 1.8 μg/ml) and a higher degree of synergy compared to any triple combination were seen. The mean DRIs of the quadruple combination were approximately twice that of the most synergistic triple combination. We conclude that human MAbs targeting different HIV-1 envelope glycoprotein epitopes exhibit strong synergy when used in combination, a fact that could be exploited clinically for passive immunoprophylaxis against HIV-1.     

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

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.     

In vivo efficacy of human immunodeficiency virus neutralizing antibodies: estimates for protective titers

The definition of plasma neutralizing antibody titers capable of controlling human immunodeficiency virus (HIV) infection in vivo is considered a critical step in vaccine development. Here we provide estimates for effective neutralization titers by assessing samples from a recent passive immunization trial with the neutralizing monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 using an analytic strategy that dissects the contributions of these MAbs to the total neutralization activity in patient plasma. Assessment of neutralization activities for six responding patients with partial or complete control of viremia during the MAb treatment and for the eight nonresponding patients revealed a significant difference between these groups: Among responders, MAb-mediated activity exceeded the autologous neutralization response by 1 to 2 log units (median difference, 43.3-fold), while in the nonresponder group, the autologous activity prevailed (median difference, 0.63-fold). In order to reach a 50% proportion of the responders in our study cohort, MAb neutralizing titers higher than 1:200 were required based on this analysis. The disease stage appears to have a significant impact on the quantities needed, since titers above 1:1,000 were needed to reach the same effect in chronic infection. Although our analysis is based on very small sample numbers and thus cannot be conclusive, our data provide a first estimate on how in vitro-measured neutralizing antibody activity can relate to in vivo efficacy in controlling HIV infection and may therefore provide valuable information for vaccine development. Interestingly, lower neutralizing antibody levels showed an effect in acute compared to chronic infection, suggesting that in early disease stages, therapeutic vaccination may show promise. Equally, this raises hopes that a preventive vaccine could become effective at comparatively lower neutralizing antibody titers.     

Nonneutralizing antibodies to the CD4-binding site on the gp120 subunit of human immunodeficiency virus type 1 do not interfere with the activity of a neutralizing antibody against the same site

We have investigated whether nonneutralizing monoclonal antibodies (MAbs) to the gp120 subunit of the envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 (HIV-1) can interfere with HIV-1 neutralization by another anti-gp120 MAb. We used neutralizing (b12) and nonneutralizing (205-42-15, 204-43-1, 205-46-9) MAbs to the epitope cluster overlapping the CD4-binding site (CD4BS) on gp120. All the MAbs, neutralizing or otherwise, cross-competed for binding to monomeric gp120, indicating the close topological proximity of their epitopes. However, the nonneutralizing CD4BS MAbs did not interfere with the neutralization activity of MAb b12. In contrast, in a binding assay using oligomeric Env expressed on the surface of Env-transfected cells, the nonneutralizing MAbs did partially compete with b12 for Env binding. The surface of Env-transfected cells contains two categories of binding site for CD4BS MAbs. One type of site is recognized by both b12 and nonneutralizing CD4BS MAbs; the other is recognized by only b12. Binding assays for Env-gp120 interactions based on the use of monomeric gp120 or Env-transfected cells do not predict the outcome of HIV-1 neutralization assays, and they should therefore be used only with caution when gauging the properties of anti-Env MAbs.     

A structure-based approach to a synthetic vaccine for HIV-1

The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --> O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with >1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.