Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in mumps virus hamster encephalitis: analysis of mutants selected with monoclonal antibodies.

With the aid of monoclonal antibodies directed against a specific site on the hemagglutinin-neuraminidase surface glycoprotein, four mutants of the Kilham neurotropic strain of mumps virus were isolated. All four mutants had increased neuraminidase activity. Two mutants (M10 and M12) lost their hemagglutination capacity with human O erythrocytes but retained their ability to agglutinate guinea pig erythrocytes at 4 degrees C. A third mutant (M11) showed a change in the molecular weight of the hemagglutinin-neuraminidase glycoprotein. These three mutants (M10, M11, and M12) showed unaltered capacity to infect tissue cultures and to cause encephalitis in newborn hamsters. A fourth mutant (M13) retained its hemagglutination activity and capacity to infect Vero cell cultures but showed significantly lower neurovirulence in the suckling hamster brain than did the parental Kilham strain and the other three mutants. Both the number of infected neurons and the amount of infectious virus in the brain was reduced. On the other hand, there were no apparent differences in the occurrence of viral antigen in ependymal cells, indicating a selective change in affinity for neurons in the brain. These results suggest that certain changes in the hemagglutinin-neuraminidase glycoprotein may lead to an alteration of the neuropathogenicity of the Kilham strain of mumps virus.     

Antibody to the E3 glycoprotein protects mice against lethal venezuelan equine encephalitis virus infection

Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.     

Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection.

Five hybridomas that secrete monoclonal antibodies which neutralize the infectivity of lactate dehydrogenase-elevating virus (LDV) were isolated from BALB/c mice primed with Formalin-inactivated LDV. Competition analyses indicated that all five neutralizing monoclonal antibodies recognize contiguous, if not identical, epitopes on the envelope glycoprotein of LDV (VP-3) which are not recognized by nonneutralizing VP-3-specific monoclonal antibodies isolated from the same fusion. Despite the presence of neutralizing activity, polyclonal anti-LDV antibodies obtained from persistently infected mice did not compete for binding to LDV with four of the five neutralizing monoclonal antibodies tested. The results indicate that the envelope glycoprotein of LDV possesses a major neutralizing epitope which is poorly recognized, if at all, by mice during a natural infection but is rendered immunogenic by Formalin inactivation of the virus. The epitope was also not immunogenic in a rabbit, since its polyclonal LDV-neutralizing antibodies did not inhibit binding of the mouse monoclonal antibodies to LDV. Passive immunization with the neutralizing monoclonal antibodies did not protect mice from LDV infection and did not alter the course of infection. Neutralizing monoclonal antibodies have been used to select a neutralization escape variant by a novel combination of in vitro and in vivo isolation.     

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.     

Conserved epitopes on the hemagglutinin-neuraminidase proteins of human and bovine parainfluenza type 3 viruses: nucleotide sequence analysis of variants selected with monoclonal antibodies.

We have previously identified 11 epitopes located in two topologically nonoverlapping antigenic sites (A and B) and a third bridging site (C) on the human type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) glycoprotein by using monoclonal antibodies (MAbs) which inhibit hemagglutination and virus infectivity (K. L. Coelingh, C. C. Winter, and B. R. Murphy, Virology 143:569-582, 1985). We have identified three additional antigenic sites (D, E, and F) on the HN molecule by competitive-binding assays of anti-HN MAbs which have no known biological activity. Epitopes in sites A, D, and F are conserved on the bovine PIV3 HN glycoprotein and also among a wide range of human isolates. The dideoxy method was used to identify nucleotide substitutions in the HN genes of antigenic variants selected with neutralizing MAbs representing epitopes in site A which are shared by human and bovine PIV3. The deduced amino acid substitutions in the variants were located in separate hydrophilic stretches of HN residues which are conserved in the primary structures of the HN proteins of both human and bovine PIV3 strains.     

Chromosomal sites of integration of simian virus 40 DNA sequences mapped by in situ hybridization in two transformed hybrid cell lines.

The neutralizing characteristics of monoclonal antibodies directed to four antigenic sites on the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus were determined. Neutralization by each antibody resulted in a persistent fraction of nonneutralized virus which varied from 1 to 17% depending on the hemagglutinin-neuraminidase site recognized, but not on the antibody. The addition of antibodies to all four sites on the hemagglutinin-neuraminidase glycoprotein was required to give a level of neutralization comparable with that obtained with polyclonal mouse antiserum. The high persistent fractions were not due to viral aggregates, a high level of variants in the virus stock, the use of insufficient antibody, low antibody avidity, or an effect peculiar to the use of the chicken cells as host. The addition of rabbit anti-mouse immunoglobulin to the persistent fraction left by any of the antibodies resulted in a further reduction in infectivity, often by as much as two logs. Thus, some viral particles are capable of binding antibody while retaining their infectivity. The implications of these findings to the mechanism of neutralization are discussed.     

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.     

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.     

Elucidation of the topography and determination of the protective epitopes on the E glycoprotein of Saint Louis encephalitis virus by passive transfer with monoclonal antibodies

We have identified and characterized eight antigenic epitopes on the 53,000 dalton envelope (E) glycoprotein of Saint Louis encephalitis (SLE) virus by using monoclonal antibodies. One of these epitopes (E-1c) encoded for the type-specific biologic functions of hemagglutination (HA) and neutralization (N). Injection of 50 ng of anti-E-1c antibody protected the majority of mice from peripheral challenge with 100 i.p. LD50 of SLE virus. Similar levels of protection with antibodies specific for other epitopes usually required greater than or equal to 1000-fold additional antibody. Attempts to block N or protection at the E-1c antigenic domain by using antibody to several other SLE epitopes that strongly competed for the E-1c site were unsuccessful. Enhancement of protection was observed with mixtures of the more cross-reactive antibodies. The E-1c antibody was also effective in abrogating SLE virus replication until neural invasion occurred. On the basis of these findings, the topologic arrangement and function of the eight SLE E glycoprotein epitopes on the virion spike is proposed.     

Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization.

Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N- and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define carbohydrate structures expressed by the viral envelope glycoprotein gp120, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both.     

Establishment and characterization of monoclonal antibodies against Chuzan virus K-47

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID₅₀/ml. Therefore, these monoclonal antibodies can evantually predict the virus infection to the animals before their sideration.     

Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift.

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.     

Protection against lethal measles virus infection in mice by immune-stimulating complexes containing the hemagglutinin or fusion protein.

The importance of each of the two surface glycoproteins of measles virus in active and passive immunization was examined in mice. Infected-cell lysates were depleted of either the hemagglutinin (H) or fusion (F) glycoprotein by using multiple cycles of immunoaffinity chromatography. The products were used to prepare immune-stimulating complexes (iscoms) containing either F or H glycoprotein. Such complexes are highly immunogenic, possibly as a result of effective presentation of viral proteins to the immune system [B. Morein, B. Sundquist, S. H?glund, K. Dalsgaard, and A. Osterhaus, Nature (London) 308:457-460, 1984]. Groups of 3-week-old BALB/c mice were inoculated with the iscom preparations. All animals developed hemolysis-inhibiting antibodies, whereas only sera of animals immunized with the iscoms containing the H glycoprotein had hemagglutination-inhibiting antibodies. Sera from animals immunized with the H or F preparation only precipitated the homologous glycoprotein in radioimmune precipitation assays. The immunized animals were challenged with a lethal dose of the hamster neurotropic variant of measles virus. Of the 7-week-old animals in the nonimmunized control group, 50% died within 10 days after challenge. No animals in the immunized groups showed symptoms of disease throughout the observation period of 3 months. Passive administration of anti-H monoclonal antibodies gave full protection against the 100% lethal acute infection with the hamster neurotropic variant of measles virus in newborn mice, whereas anti-F monoclonal antibodies failed to protect the animals. This study emphasizes that both H and F glycoproteins need to be considered in the development of measles virus subunit vaccines.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Antigenic analysis of equine infectious anemia virus (EIAV) variants by using monoclonal antibodies: epitopes of glycoprotein gp90 of EIAV stimulate neutralizing antibodies.

Monoclonal antibodies produced against the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV), a lentivirus, were studied for reactivity with the homologous prototype and 16 heterologous isolates. Eighteen hybridomas producing monoclonal antibodies (MAbs) were isolated. Western blot (immunoblot) analyses indicated that 10 were specific for the major envelope glycoprotein (gp90) and 8 for the transmembrane glycoprotein (gp45). Four MAbs specific to epitopes of gp90 neutralized prototype EIAV infectivity. These neutralizing MAbs apparently reacted with variable regions of the envelope gp90, as evidenced by their unique reactivity with the panel of isolates, suggesting recognition of at least three different neutralization epitopes. The conformation of these epitopes appears to be continuous, as they resisted treatment with sodium dodecyl sulfate and reducing reagents. Monoclonal antibodies that reacted with conserved epitopes on gp90 or gp45 failed to neutralize EIAV. Our data also demonstrated that there was a large spectrum of possible EIAV serotypes and confirmed that antigenic variation occurs with high frequency in EIAV. Moreover, the data showed that variation is a rapid and random process, as no pattern of variant evolution was evident by comparison of 13 isolates from parallel infections. These results represent the first production of neutralizing MAbs specific for a lentivirus glycoprotein and document alterations in one or more neutralization epitopes of the major surface glycoprotein among sequential isolates of EIAV recovered during persistent infection.     

Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes

Varicella-zoster virus (VZV) codes for approximately eight glycosylated polypeptides in infected cell cultures and in virions. To determine the number of serologically distinct glycoprotein gene products encoded by VZV, we have developed murine monoclonal antibodies to purified virions. Of 10 monoclonal antibodies which can immunoprecipitate intracellular VZV antigens and virion glycoproteins, 1 (termed gA) reacted with gp105, 1 (termed gB) reacted with gp115 (intracellular only), gp62, and gp57, and 8 (termed gC) reacted with gp92, gp83, gp52, and gp45. The anti-gA monoclonal antibody neutralized VZV infectivity in the absence of complement. All eight anti-gC monoclonal antibodies neutralized only in the presence of complement. An anti-gB monoclonal antibody obtained from another laboratory also neutralizes in the absence of complement. Since the above reactivities account for all major detectable VZV glycoprotein species, the data strongly suggest that VZV has three major glycoprotein genes which encode glycosylated polypeptides with neutralization epitopes.     

Antibody responses of humans and nonhuman primates to individual antigenic sites of the hemagglutinin-neuraminidase and fusion glycoproteins after primary infection or reinfection with parainfluenza type 3 virus.

An unusual feature of human parainfluenza virus type 3 (PIV3) is ita ability to cause reinfection with high efficiency. The antibody responses of 45 humans and 9 rhesus monkeys to primary infection or subsequent reinfection with PIV3 were examined to identify deficiencies in host immunologic responses that might contribute to the ability of the virus to cause reinfection with high frequency. Antibody responses in serum were tested by using neutralization and hemagglutination inhibition (HI) assays and a monoclonal antibody blocking immunoassay able to detect antibodies to epitopes within six antigenic sites on the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein and eight antigenic sites on the fusion (F) protein. Primary infection of seronegative infants or children with PIV3 stimulated strong and rather uniform HI and neutralizing antibody responses. More than 90% of the individuals developed antibodies to four of the six HN antigenic sites (including three of the four neutralization sites), but the responses to F antigenic sites were of lesser magnitude and varied considerably from person to person. Young infants who possessed maternally derived antibodies in their sera developed lower levels and less frequent HI, neutralizing, and antigenic site-specific responses to the HN and F glycoproteins than did seronegative infants and children. In contrast, children reinfected with PIV3 developed even higher HI and neutralizing antibody responses than those observed during primary infection. Reinfection broadened the HN and F antigenic site-specific responses, but the latter remained relatively restricted. Adults possessed lower levels of HI, neutralizing, and antigenic site-specific antibodies in their sera than did children who had been reinfected, suggesting that these antibodies decay with time. Rhesus monkeys developed more vigorous primary and secondary antibody responses than did humans, but even in these highly responsive animals, response to the F glycoprotein was relatively restricted following primary infection. Bovine PIV3 induced a broader response to human PIV3 in monkeys than was anticipated on the basis of their known relatedness as defined by using monoclonal antibodies to human PIV3. These observations suggest that the restricted antibody responses to multiple antigenic sites on the F glycoprotein in young seronegative infants and children and the decreased responses to both the F and HN glycoproteins in young infants and children with maternally derived antibodies may play a role in the susceptibility of human infants and young children to reinfection with PIV3.     

Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies.

Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.     

Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus.

Four distinct antigenic determinants along the G2 glycoprotein encoded by the M segment RNA of the Phlebovirus Rift Valley fever virus were localized. These epitopes were defined by four monoclonal antibodies, three of which were capable of neutralizing virus infectivity; one was nonneutralizing. Immunoprecipitation by these monoclonal antibodies of either denatured or native antigen characterized the epitopes as having linear or higher order structure. Molecular cloning of G2 glycoprotein-coding sequences into a bacterial expression plasmid utilizing a beta-galactosidase fusion protein system was employed for epitope localization. A nuclease BAL 31 plasmid expression library, in which processive regions of the 3' end of the G2 glycoprotein coding sequences were deleted, allowed for approximation of the carboxy-terminal limit of the antigenic determinants. Further subcloning of limited G2 polypeptide sequences into the bacterial expression vector permitted more refined localization of the epitopes. The characteristics of the immunoreactivity of these small peptide regions (between 11 and 34 amino acids) produced in bacteria as G2-beta-galactosidase fusion proteins were similar to those of the authentic Rift Valley fever virus G2 glycoprotein. These defined antigenic determinants and their importance in virus infectivity are discussed.     

Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.