Restricted occupancy models for neutralization of HIV virions and populations

HIV virions infect cells by attaching to target cell receptors, fusing membranes with the cell and by finally releasing their genetic material into the target cells. Antibodies can hinder the infection by attaching to the HIV envelope glycoprotein trimers before or during attachment. The exact mechanisms and the quantitative requirements of antibody neutralization are still debated. Recently, the number of antibodies rendering one trimer non-functional, called stoichiometry of (trimer) neutralization, was studied with mathematical models. Here we extend this theoretical framework to calculate the stoichiometries of neutralizing a single virion and a whole virion population. We derive mathematical equations for antibody neutralization based on restricted occupancy theory. Additionally we simulate these processes when a direct calculation is not possible. We find that the number of trimers needed for cell entry and the number of antibodies neutralizing one trimer strongly influence the mean number of antibodies needed for virion and population neutralization. Further we show that the mean number of antibodies needed to neutralize a virion population exceeds the product of the number of virions in the population and the mean number of antibodies needed to neutralize one virion.     

A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this "multiple-hit" perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus "breathing" in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization.     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.     

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.     

Immunodominance and functional activities of antibody responses to inactivated West Nile virus and recombinant subunit vaccines in mice

Factors controlling the dominance of antibody responses to specific sites in viruses and/or protein antigens are ill defined but can be of great importance for the induction of potent immune responses to vaccines. West Nile virus and other related important human-pathogenic flaviviruses display the major target of neutralizing antibodies, the E protein, in an icosahedral shell at the virion surface. Potent neutralizing antibodies were shown to react with the upper surface of domain III (DIII) of this protein. Using the West Nile virus system, we conducted a study on the immunodominance and functional quality of E-specific antibody responses after immunization of mice with soluble protein E (sE) and isolated DIII in comparison to those after immunization with inactivated whole virions. With both virion and sE, the neutralizing response was dominated by DIII-specific antibodies, but the functionality of these antibodies was almost four times higher after virion immunization. Antibodies induced by the isolated DIII had an at least 15-fold lower specific neutralizing activity than those induced by the virion, and only 50% of these antibodies were able to bind to virus particles. Our results suggest that immunization with the tightly packed E in virions focuses the DIII antibody response to the externally exposed sites of this domain which are the primary targets for virus neutralization, different from sE and isolated DIII, which also display protein surfaces that are cryptic in the virion. Despite its low potency for priming, DIII was an excellent boosting antigen, suggesting novel vaccination strategies that strengthen and focus the antibody response to critical neutralizing sites in DIII.     

Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature.

Antisera were raised against peptide sequences that are normally internal in the poliovirus virion. These antisera contain neutralizing activity, but this neutralizing activity is dependent on coincubation of the virus and antisera at 37 degrees C. Immunoprecipitation analyses demonstrate that the neutralization is due to exposure of these normally internal sequences at 37 degrees C and subsequent antibody binding. Exposure of these sequences is reversible. These data demonstrate that the poliovirus particle is a dynamic entity that is capable of undergoing conformational alterations at physiological temperatures. This conformational flexibility provides an explanation for earlier observations of virus neutralization by antibodies to internal epitopes which can be accommodated within the framework of existing models for antibody-mediated neutralization of viral infectivity. Analogies between the sequences which are reversibly exposed at 37 degrees C with those which are irreversibly exposed upon receptor binding suggest that the observed conformational dynamics also may play a role in cell entry.     

Dissection of Antibody Specificities Induced by Yellow Fever Vaccination

The live attenuated yellow fever (YF) vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III) and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity) as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific factors on immunodominance in humoral immune responses.     

Studies on the neutralization of herpes simplex virus. VIII. Significance of viral sensitization for inactivation by complement.

Early and late IgG of rabbits immunized with herpes virus showed, respectively, 8-fold and 2-fold enhancement of neutralization endpoint in the presence of complement (C). Kinetic curve experiments employing an appropriate amount of virus revealed that both neutralization and sensitization followed first-order reaction, and each IgG possessed a certain range of concentration where neutralization was negligible while sensitization was marked. Dose responses of neutralization and sensitization velocities demonstrated that the C enhancement of late IgG was about 7-fold and that of early IgG more than 20-fold. These facts suggested that the IgGs contained two different entities of complement-requiring (CRN) and non-requiring neutralizing (N) antibodies at different proportions, only the former being responsible for sensitization. The different CRN: N ratios obtained by the endpoint and kinetic methods may mean either that the two antibodies differ in avidity for the virus or that the number of critical sites per virion for CRN antibody is greater than that for N antibody. In this interpretation, sensitization by CRN antibody as well as neutralization by N antibody is thought to result from attachment of a single antibody molecule to the viral critical site. Alternative explanations, ascribing the mechanism of neutralization to steric hindrance of critical sites or to multiple hit of those sites by antibody, were denied by analyses of the present data.     

Stoichiometry of antibody neutralization of human immunodeficiency virus type 1

The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.     

The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection

Antibody binding to the icosahedral arrangement of envelope proteins on the surface of flaviviruses can result in neutralization or enhancement of infection. We evaluated how many antibodies must bind to a given epitope on West Nile virus (WNV) to achieve neutralization. The most potent monoclonal antibodies (mAbs) block infection at concentrations that result in low occupancy of accessible sites on the virion, with neutralization occurring when as few as 30 of 180 envelope proteins are bound. In contrast, weakly neutralizing mAbs recognize fewer sites on the virion and require almost complete occupancy to inhibit WNV infection. For all mAbs studied, enhancement of infection is possible in cells bearing activating Fc-gamma receptors when the number of mAbs docked to the virion is not sufficient for neutralization. Thus, neutralization is best described by a model requiring "multiple hits" with the cumulative functional outcome determined by interplay between antibody affinity and epitope accessibility.     

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.     

Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response.

Antisera raised against Rauscher leukemia virus (R-MuLV) contain a preponderance of antibodies against glycoprotein gp70 that are dependent on the presence of carbohydrate side chains for reactivity, as judged by immunoprecipitation or Western blotting. However, the majority of neutralizing antibodies were not dependent on the presence of carbohydrate, as indicated by (i) the ability of deglycosylated R-MuLV to adsorb neutralizing antibody from sera as efficiently as glycosylated R-MuLV and (ii) the ability of deglycosylated R-MuLV to induce neutralizing antibody responses when injected into rabbits. Moreover, a faster response was obtained with deglycosylated R-MuLV than with untreated control virus in the latter experiments. The results indicate that the neutralizing antibodies are a discrete subpopulation of the total antibody response. Furthermore, the carbohydrate moieties appear to afford protection to the virion during infection, rather than serve as a target for neutralization.     

Postentry neutralization of adenovirus type 5 by an antihexon antibody

Antibodies against hexon, the major coat protein of adenovirus (Ad), are an important component of the neutralizing activity in serum from naturally infected humans and experimentally infected animals. The mechanisms by which antihexon antibodies neutralize the virus have not been defined. As a model system, murine monoclonal antibodies raised against Ad type 5 (Ad5) were screened for antihexon binding and neutralization activity; one monoclonal antibody, designated 9C12, was selected for further characterization. The minimum ratio of 9C12 to Ad5 required for neutralization was 240 antibody molecules per virus particle, or 1 antibody per hexon trimer. Analysis of antibody-virus complexes by dynamic light scattering and negative-stain electron microscopy (EM) showed that the virus particles were coated with electron-dense material but not aggregated at neutralizing ratios. Cryo-EM image reconstruction of the antibody-virus complex showed that the surface of the virus particle was covered by a meshwork of 9C12 antibody density, consistent with bivalent binding at multiple sites. Confocal analysis revealed that viral attachment, cell entry, and intracellular transport to the nuclear periphery still occur in the presence of neutralizing levels of 9C12. A model is presented for neutralization of Ad by an antihexon antibody in which the hexon capsid is cross-linked by antibodies, thus preventing virus uncoating and nuclear entry of viral DNA.     

Rubella virus antigens: localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies.

Monoclonal antibodies (MAbs) against the rubella virion were used to locate epitopes involved in hemagglutination and neutralization. The MAbs exhibiting high-level hemagglutination-inhibiting activity were shown by Western blot analysis to be specific for the E1 polypeptide; this is consistent with the presence of the hemagglutinin on the E1 polypeptide. Some of the E1-specific MAbs also neutralized viral infectivity. However, hemagglutination-inhibiting activity and neutralizing activity did not always correlate. Three distinct functional epitopes were identified on the E1 polypeptide by competition analyses: one which reacted with MAbs with high-level hemagglutination-inhibiting activity and with neutralizing activity, one which reacted with MAbs with low-level hemagglutination-inhibiting activity and with neutralizing activity, and one which reacted with MAbs with only hemagglutination-inhibiting activity. A MAb specific for the E2 polypeptide exhibited neutralizing activity. This E2-specific MAb and two E1-specific MAbs with neutralizing activity were capable of precipitating intact virus which indicates that at least three epitopes involved in neutralization are accessible on the surface of the virion.     

Syngeneic monoclonal anti-idiotype antibodies that bear the internal image of a human cytomegalovirus neutralization epitope

Two glycoproteins have been identified on human CMV that induce neutralizing antibody; an 86,000-Da glycoprotein and a 130,000-, 92,000-, and 50,000-Da glycoprotein coimmunoprecipitating complex that appears to be the gB homologue of HSV. We have produced syngeneic monoclonal anti-Id antibodies (mAb2) of the IgM isotype to a CMV-neutralizing monoclonal antibody (mAb1) that is known to bind to the 86,000-Da glycoprotein on the virion envelope. These mAb2 bear the internal image of the original viral antigen as shown by their ability to 1) recognize an interspecies idiotype in CMV-positive human antisera, 2) block mAb1 binding to CMV antigen, and 3) block CMV neutralization by mAb1 in vitro. Immunization of mice with both of these affinity chromatography-purified mAb2 stimulated the production of anti-anti-Id monoclonal antibodies (which we termed mAb3), which bound to the mAb2 by ELISA and neutralized CMV infectivity.     

Molecular basis for linkage of a continuous and discontinuous neutralization epitope on the structural polypeptide VP2 of poliovirus type 1.

We obtained neutralizing monoclonal antibodies against a continuous neutralization epitope on VP2 of poliovirus type 1 strain Mahoney by using a combined in vivo-in vitro immunization procedure. The antibody-binding site was mapped to amino acid residues within the peptide segment (residues 164 through 170) of VP2 by competition with synthetic peptide and sequencing of resistant mutants. Cross-neutralization of these mutants with another neutralizing monoclonal antibody revealed a linkage of the continuous epitope and a discontinuous neutralization epitope involving both loops of the double-loop structure of VP2 at the twofold axis on the surface of the virion.     

Time frames for neutralization during the human immunodeficiency virus type 1 entry phase, as monitored in synchronously infected cell cultures

Characterization of the neutralizing interaction between antibody and virus is hindered by the nonsynchronized progression of infection in cell cultures. Discrete steps of the viral entry sequence cannot be discerned, and thus, the mode of antibody-mediated interference with virus infectivity remains undefined. Here, we magnetically synchronize the motion and cell attachment of human immunodeficiency virus type 1 (HIV-1) to monitor the progression of neutralization, both in solution and following virus attachment to the cell. By simultaneous transfer of all viral particles from reaction solution with antibody to the cell-bound state, the precise rate of neutralization of cell-free virus could be determined for each antibody. HIV-1 neutralization by both monoclonal and polyclonal antibody preparations followed distinct pseudo-first-order kinetics. For all antibodies, cell types, and HIV-1 strains examined, postattachment interference served a major role in the neutralizing effect. To monitor the progression of postattachment interference, we synchronized the entry process at initiation and measured the escape of cell-bound virus from antibody. We found that different antibodies neutralized the virus over different time frames during the entry phase. Virus was observed to progress through a sequence of shifting sensitivities to different antibodies during entry, suggested here to correlate with the exposure time of the target epitope on receptor-activated viral envelope proteins. Thus, by monitoring the progression of HIV-1 entry under synchronized conditions, we identify a new and significant determinant of antibody neutralization capacity, namely, the time frames for neutralization during the course of the viral entry phase.     

Design of Escherichia coli-Expressed Stalk Domain Immunogens of H1N1 Hemagglutinin That Protect Mice from Lethal Challenge

The hemagglutinin protein (HA) on the surface of influenza virus is essential for viral entry into the host cells. The HA1 subunit of HA is also the primary target for neutralizing antibodies. The HA2 subunit is less exposed on the virion surface and more conserved than HA1. We have previously designed an HA2-based immunogen derived from the sequence of the H3N2 A/HK/68 virus. In the present study, we report the design of an HA2-based immunogen from the H1N1 subtype (PR/8/34). This immunogen (H1HA0HA6) and its circular permutant (H1HA6) were well folded and provided complete protection against homologous viral challenge. Antisera of immunized mice showed cross-reactivity with HA proteins of different strains and subtypes. Although no neutralization was observable in a conventional neutralization assay, sera of immunized guinea pigs competed with a broadly neutralizing antibody, CR6261, for binding to recombinant Viet/04 HA protein, suggesting that CR6261-like antibodies were elicited by the immunogens. Stem domain immunogens from a seasonal H1N1 strain (A/NC/20/99) and a recent pandemic strain (A/Cal/07/09) provided cross-protection against A/PR/8/34 viral challenge. HA2-containing stem domain immunogens therefore have the potential to provide subtype-specific protection.     

Inhibition of rhinovirus attachment by neutralizing monoclonal antibodies and their Fab fragments.

Previous molecular and immunological studies have mapped four neutralization sites on human rhinovirus type 14 (B. Sherry, A. G. Mosser, R. J. Colonno, and R. R. Rueckert, J. Virol. 57:246-257, 1986). Eight monoclonal antibodies, one pair for each of the four target sites and all belonging to a single isotype, immunoglobulin G2a, were studied under conditions which resulted in 95% neutralization of infectious viral particles. All eight antibodies shifted the isoelectric point of virions from 6.7 to much more acidic forms, ranging from pI 1.8 to 3.2. In addition, antibodies targeted against three of the four neutralization sites caused significant aggregation of virions under the neutralization conditions employed. Aggregation could be reversed by digesting virus-antibody complexes with papain. Following papain digestion, the acidic pIs of three of the neutralized virus preparations returned to neutral and infectivity was restored. Membrane-binding assays with virus neutralized with a nonaggregating antibody showed a dose-related inhibition of virus attachment to cellular receptors. Purified Fab fragments at a 13- to 61-fold-higher concentration than intact antibodies caused a comparable isoelectric shift, neutralized virions in the absence of aggregation, and interfered with attachment of virions to host cell receptors in a membrane-binding assay. These findings suggest that neutralizing antibodies interfere with the attachment of rhinoviruses to cellular receptors and that bivalent attachment of antibody is not a prerequisite for neutralization.     

Identification of a new neutralization antigenic site on poliovirus coat protein VP2

Major neutralization antigenic sites have been previously mapped by us on VP1, the largest capsid protein of poliovirus type 1. Here we report the first identification of the primary sequence of a neutralization antigenic site on capsid protein VP2. Inspection of the amino acid sequence of VP2 led to the selection and synthesis of a peptide (n = 12) that, after linking to a carrier protein, induced an antiviral neutralizing antibody response in rabbits. The response was augmented by a single subsequent inoculation of intact virus; thus, the peptide was also capable of priming the production of neutralizing antibodies. These antibodies were directed only against the site specified by the synthetic peptide. Although the VP2-specific neutralization antigenic site appears not to be strongly immunogenic in the intact virion, it can nevertheless contribute to neutralization of poliovirus. This observation may be important for the development of peptide vaccines.