Determination of the protective epitopes on the glycoproteins of Venezuelan equine encephalomyelitis virus by passive transfer of monoclonal antibodies

We have previously characterized seven unique antigenic epitopes on the two envelope glycoproteins of the Venezuelan equine encephalomyelitis (VEE) virus vaccine strain, TC-83, by using monoclonal antibodies. The in vitro function of virus neutralization was primarily associated with one epitope on the gp56 (gp56c). To determine which epitopes were important in protecting animals from VEE infection, purified monoclonal antibodies were inoculated i.v. into 3-wk-old Swiss mice. Twenty-four hours later these animals were challenged i.p. with 100 IPLD50 of virulent VEE virus (Trinidad donkey). High-avidity anti-gp56c, anti-gp50b, anti-gp50c, and anti-gp50d monoclonal antibodies protected animals from virus challenge. Rabbit antisera to the gp56 and the gp50 glycoproteins were also effective in protecting mice from challenge with virulent VEE virus. Less antibody was needed to protect animals if the antibody was directed against the critical neutralization site. Less avid antibodies to the gp56c and gp50b epitopes demonstrated little or no protection in vivo. Protection, therefore, appeared to be a function of the passive antibody's specificity, avidity, and ability to bind to virion antigenic determinants topologically proximal to the critical neutralization site.     

Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge.

Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.     

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.     

A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.     

Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice

Neuroadapted Sindbis virus (NSV) causes acute encephalitis and paralyzes and kills adult mice unless they are treated with primary immune serum after infection. To study the nature and specificity of curative antibodies, we gave mice 30 different monoclonal antibodies (MAbs) against Sindbis virus (SV) 24 h after lethal intracerebral inoculation of NSV. By the time of MAb treatment, NSV replication in the brain had been well established (7.5 X 10(7) PFU/g). Seventeen MAbs directed against multiple biological domains on the NSV E1 and E2 envelope glycoproteins prevented paralysis and death. Anticapsid MAbs failed to protect. Altogether, 15 of 17 curative MAbs either neutralized NSV infectivity or lysed NSV-infected cells with complement, but neither ability was necessary or sufficient to guarantee recovery. All 5 protective anti-E2 MAbs neutralized NSV infectivity; 6 of 10 protective anti-E1 MAbs neutralized NSV; 4 did not. Plaque assay or immunohistochemical staining showed that neutralizing and nonneutralizing curative MAbs decreased NSV in the brain, brainstem, and spinal cord. Despite high neutralization titers, hyperimmune anti-SV and anti-NSV mouse sera prevented only 6 and 30% of deaths, respectively, while primary immune sera prevented 50 (SV) and 90% (NSV) of deaths. Secondary intravenous immunization with a live virus apparently diminished, obscured, or failed to boost a class of protective antibodies. When separate mouse groups were given these 30 MAbs 24 h before lethal intracerebral inoculation of NSV, a slightly different set of 17 neutralizing or nonneutralizing anti-E1 and anti-E2 antibodies protected. Two nonneutralizing MAbs and hyperimmune anti-SV serum, which had failed to promote recovery, prophylactically protected 100% of the mice. The antibody requirements or mechanisms of prophylaxis and recovery may differ.     

Monoclonal antibody-defined epitope map of expressed rubella virus protein domains.

An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.     

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.     

An antibody- and synthetic peptide-defined rubella virus E1 glycoprotein neutralization domain.

We previously described a monoclonal antibody (MAb) library generated by infecting BALB/c mice with rubella virus (RV) and selected by an enzyme-linked immunosorbent assay (ELISA) using purified virion targets. Plasmid pARV02-01, which expresses the fusion protein RecA1-35-GIGDLGSP-E1(202)-E1(283)-GDP-LacZ9-1015 in Escherichia coli, was shown to be a ligand for MAbs E1-18 and E1-20 (J. S. Wolinsky, M. McCarthy, O. Allen-Cannady, W. T. Moore, R. Jin, S. N. Cao, A. Lovett, and D. Simmons, J. Virol. 65:3986-3994, 1991). Both of these MAbs neutralize RV infectivity. A series of five overlapping synthetic peptides was made to further explore the requirements of this MAb binding domain. One of these peptides (SP15; E1(208) to E1(239)) proved an effective ligand for both MAbs in the ELISA. Stepwise synthesis of SP15 defined the minimal amino-terminal requirement for binding MAb E1-18 as E1(221) and that of MAb E1-20 as E1(223); the minimal carboxyl-terminal requirement is uncertain but does not exceed E1(239). Immunization of mice and rabbits with SP15 induced polyvalent antibody reactive with SP15, with other overlapped and related but not unrelated synthetic peptides, and with RV. The rabbit anti-SP15 antibody showed neutralization activity to RV similar to that of MAbs E1-18 and E1-20 but lacked hemagglutination inhibition activity. These data define a neutralization domain on E1 and suggest that the RV epitopes conserved by SP15 may be critical for protective host humoral immune responses.     

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.     

Binding of anti-membrane-proximal gp41 monoclonal antibodies to CD4-liganded and -unliganded human immunodeficiency virus type 1 and simian immunodeficiency virus virions

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.     

Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar^−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.     

Poorly neutralizing cross-reactive antibodies against the fusion loop of West Nile virus envelope protein protect in vivo via Fcgamma receptor and complement-dependent effector mechanisms

The human antibody response to flavivirus infection is dominantly directed against a cross-reactive epitope on the fusion loop of domain II (DII-FL) of the envelope (E) protein. Although antibodies against this epitope fail to recognize fully mature West Nile virus (WNV) virions and accordingly neutralize infection poorly in vitro, their functional properties in vivo remain less well understood. Here, we show that while passive transfer of poorly neutralizing monoclonal antibodies (MAb) and polyclonal antibodies against the DII-FL epitope protect against lethal WNV infection in wild-type mice, they fail to protect mice lacking activating Fcγ receptors (FcγR) and the complement opsonin C1q. Consistent with this, an aglycosyl chimeric mouse-human DII-FL MAb (E28) variant that lacks the ability to engage FcγR and C1q also did not protect against WNV infection in wild-type mice. Using a series of immunodeficient mice and antibody depletions of individual immune cell populations, we demonstrate that the nonneutralizing DII-FL MAb E28 does not require T, B, or NK cells, inflammatory monocytes, or neutrophils for protection. Rather, E28 treatment decreased viral load in the serum early in the course of infection, which resulted in blunted dissemination to the brain, an effect that required phagocytic cells, C1q, and FcγRIII (CD16). Overall, these studies enhance our understanding of the functional significance of immunodominant, poorly neutralizing antibodies in the polyclonal human anti-flavivirus response and highlight the limitations of current in vitro surrogate markers of protection, such as cell-based neutralization assays, which cannot account for the beneficial effects conferred by these antibodies.     

Protection against lethal viral infection by neutralizing and nonneutralizing monoclonal antibodies: distinct mechanisms of action in vivo.

Monoclonal antibodies (MAb) reactive with the glycoprotein of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were used to protect mice against lethal infection. MAb which reacted with a number of distinct epitopes and which could neutralize the virus in vitro could also protect against infection in vivo. MAb which could not neutralize the virus in vitro but which were specific for the glycoprotein of a single serotype were also able to protect mice against lethal VSV challenge. Interestingly, a group of MAb which cross-reacted with the glycoproteins of VSV-Ind and VSV-NJ could passively protect against challenge with either serotype. It was shown that as early as 2 h after infection, neither neutralizing nor nonneutralizing MAb could protect. Nonneutralizing MAb were found to be less effective at in vivo protection than neutralizing MAb. Furthermore, nonneutralizing MAb demonstrated a much lower binding efficiency to intact virions than did neutralizing MAb. These observations, plus the fact that the nonneutralizing MAb could lyse virus-infected cells in the presence of complement, suggested that in vivo protection by these antibodies may involve cell-associated viral determinants. To compare the mechanisms by which neutralizing and nonneutralizing MAb protected in vivo, F(ab')2 fragments were used in protection experiments. Although the F(ab')2 of a neutralizing MAb was still able to protect animals lethal virus challenge, the F(ab')2 of a cross-reactive nonneutralizing MAb was unable to do so. The reactivity of nonneutralizing MAb with virions and the apparent necessity of an intact Fc portion for protection further distinguish these antibodies from those MAb that are able to neutralize VSV solely by binding to the glycoprotein.     

Resistance to Alpha/Beta Interferons Correlates with the Epizootic and Virulence Potential of Venezuelan Equine Encephalitis Viruses and Is Determined by the 5′ Noncoding Region and Glycoproteins

We compared the alpha/beta interferon (IFN-α/β) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-α/β resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5′ noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.     

Intratypic variations in neutralizable epitopes among herpes simplex virus type 2 isolates.

Intratypic variation among 94 isolates of herpes simplex virus type 2 (HSV-2) was investigated using 4 different monoclonal antibodies (MAbs). By neutralization test, these MAbs appeared to be directed to at least 2 distinct epitopes on the viral glycoprotein D (gD), i.e., 6G6.G9 and 6E8.F11 which did not require complement (C-MAb) and gD-105 and gD-110 whose neutralizing activities could be enhanced by complement (C+MAb). The C-MAb pairs each separately could detect significant intratypic variations among the isolates. Whether these variations also existed in the gD epitope(s) recognized by C+MAbs remains to be elucidated. The results suggested that intratypic variation occurred on at least one of the neutralizable (thus related to protective immunity) epitopes on gD of HSV-2.     

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.     

Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.     

Critical epitopes in transmissible gastroenteritis virus neutralization.

Purified transmissible gastroenteritis (TGE) virus was found to be composed of three major structural proteins having relative molecular weights of 200,000, 48,000, and 28,000. The peplomer glycoprotein was purified by affinity chromatography with the monoclonal antibody (MAb) 1D.G3. A collection of 48 MAbs against TGE virus was developed from which 26, 10, and 3 were specific for proteins E2, N, and E1, respectively. A total of 14 neutralizing MAbs of known reactivity were E2 protein specific. In addition, MAb 1B.C11, of unknown specificity, was also neutralizing. These MAbs reduced the virus titer 10(2)- to 10(9)-fold. Six different epitopes critical in TGE virus neutralization were found, all of which were conformational based on their immunogenicity and antigenicity. Only the epitope defined by MAb 1G.A7 was resistant to sodium dodecyl sulfate treatment, although it was destroyed by incubation in the presence of both the detergent and beta-mercaptoethanol. The frequency of MAb-resistant (mar) mutants selected with four MAbs (1G.A7, 1B.C11, 1G.A6, and 1E.F9) ranged from 10(-6) to 10(-7), whereas the frequency of the putative mar mutant defined by MAb 1B.B11 was lower than 10(-9). Furthermore, the epitopes defined by these MAbs and by MAbs 1H.C2 and 1A.F10, were present in 11 viral isolated with different geographical locations, years of isolation, and passage numbers (with the exception of two epitopes absent or modified in the TOY 56 viral isolate), suggesting that the critical epitopes in TGE virus neutralization were highly conserved.     

Roles and reactivities of antibodies to alphaviruses

Antibodies to alphaviruses are essential for recovery from infection. These antibodies act by interacting with the E1 and E2 glycoproteins on the virion and on the surface of infected cells. E1 epitopes are displayed primarily on infected cells and are cryptic on the virion until after exposure to acidic pH. A single neutralizing epitope (E1-c) includes E1 residue 132. Two dominant neutralizing domains have been identified on the Sindbis virus E2: E2-ab from E2-190 to 216 and E2-c from E2-62 to 159. E2-ab is likely to form a single loop and contain linear determinants while E2-c is strictly conformational involving several sites on the linear molecule. Both neutralizing and non-neutralizing E1 and E2 MAbs can protect and promote recovery from fatal encephalitis. E2 MAb can clear infectious virus from persistently infected cells by non-cytolytic process and acts synergistically with interferon.     

Properties of monospecific antibodies to the glycoprotein of western equine encephalitis virus

Monospecific (MSp-) antisera against E1 and E2 glycoproteins of western equine encephalitis (WEE) virus were prepared and examined for binding activities to whole virions, hemagglutination-inhibition (HI), neutralization (NT) and protection. Both anti-E1 and anti-E2 MSp-Abs protected mice against WEE virus challenge. A competition experiment with monoclonal antibodies showed that these MSp-antisera appear to lack the antibody population for some epitopes involved in viral neutralization.