Antigenic and structural properties of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3: sequence analysis of variants selected with monoclonal antibodies which inhibit infectivity, hemagglutination, and neuraminidase activities.

The hemagglutinin-neuraminidase (HN) gene sequence was determined for 16 antigenic variants of human parainfluenza virus type 3 (PIV3). The variants were selected by using monoclonal antibodies (MAbs) to the HN protein which inhibit neuraminidase, hemagglutination, or both activities. Each variant had a single-point mutation in the HN gene, coding for a single amino acid substitution in the HN protein. Operational and topographic maps of the HN protein correlated well with the relative positions of the substitutions. There was little correlation between the cross-reactivity of a MAb with the bovine PIV3 HN and the amount of amino acid homology between the human and bovine PIV3 HN proteins in the regions of the epitopes, suggesting that many of the epitopes are conformational in nature. Computer-assisted analysis of the HN protein predicted a secondary structure composed primarily of hydrophobic beta sheets interconnected by random hydrophilic coil structures. The HN epitopes were located in predicted coil regions. Epitopes recognized by MAbs which inhibit neuraminidase activity of the virus were located in a region which appears to be structurally conserved among several paramyxovirus HN proteins and which may represent the sialic cid-binding site of the HN molecule.     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.     

Identification of amino acids recognized by syncytium-inhibiting and neutralizing monoclonal antibodies to the human parainfluenza type 3 virus fusion protein.

Neutralizing monoclonal antibodies specific for the fusion (F) glycoprotein of human parainfluenza type 3 virus (PIV3) were used to select neutralization-resistant antigenic variants. Sequence analysis of the F genes of the variants indicated that their resistance to antibody binding, antibody-mediated neutralization or to both was a result of specific amino acid substitutions within the neutralization epitopes of the F1 and F2 subunits. Comparison of the locations of PIV3 neutralization epitopes with those of Newcastle disease and Sendai viruses indicated that the antigenic organization of the fusion proteins of paramyxoviruses is similar. Furthermore, some of the PIV3 epitopes recognized by syncytium-inhibiting monoclonal antibodies are located in an F1 cysteine cluster region which corresponds to an area of the measles virus F protein involved in fusion activity.     

Neutralization map of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus: domains recognized by monoclonal antibodies that prevent receptor recognition.

Monoclonal antibodies (MAbs) to the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus delineate seven overlapping antigenic sites which form a continuum on the surface of the molecule. Antibodies to five of these sites neutralize viral infectivity principally by preventing attachment of the virion to cellular receptors. Through the identification of single amino acid substitutions in variants which escape neutralization by MAbs to these five antigenic sites, a neutralization map of HN was constructed, identifying several residues that contribute to the epitopes recognized by MAbs which block the attachment function of the molecule. These epitopes are defined, at least in part, by three domains on HN: residues 193 to 201; 345 to 353 (which include the only linear epitope we have identified in HN); and a C-terminal domain composed of residues 494, 513 to 521, and 569. To identify HN residues directly involved in receptor recognition, each of the variants was tested for its ability to agglutinate periodate-modified chicken erythrocytes. One variant with a single amino acid substitution at residue 193 was 2.5- to 3-fold more resistant to periodate treatment of erythrocytes than the wild-type virus, suggesting that this residue influences the binding of virus to a sialic acid-containing receptor(s) on the cell surface.     

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.     

Naturally occurring human parainfluenza type 3 viruses exhibit divergence in amino acid sequence of their fusion protein neutralization epitopes and cleavage sites.

Many human parainfluenza type 3 virus (PIV3) strains isolated from children with respiratory illness are resistant to neutralization by monoclonal antibodies (MAbs) which recognize epitopes in antigenic site A or B of the fusion (F) protein of the prototype 1957 PIV3 strain. The F protein genes of seven PIV3 clinical isolates were sequenced to determine whether their neutralization-resistant phenotypes were associated with specific differences in amino acids which are recognized by neutralizing MAbs. Several clinical strains which were resistant to neutralization by site A or B MAbs had amino acid differences at residues 398 or 73, respectively. These specific changes undoubtedly account for the neutralization-resistant phenotype of these isolates, since identical substitutions at residues 398 or 73 in MAb-selected escape mutants confer resistance to neutralization by site A or B MAbs. The existence of identical changes in naturally occurring and MAb-selected neutralization-resistant PIV3 strains raises the possibility that antigenically different strains may arise by immune selection during replication in partially immune children. Three of the seven clinical strains examined had differences in their F protein cleavage site sequence. Whereas the prototype PIV3 strain has the cleavage site sequence Arg-Thr-Lys-Arg, one clinical isolate had the sequence Arg-Thr-Arg-Arg and two isolates had the sequence Arg-Thr-Glu-Arg. The different cleavage site sequences of these viruses did not affect their level of replication in either continuous simian or bovine kidney cell monolayers (in the presence or absence of exogenous trypsin or plasmin) or in the upper or lower respiratory tract of rhesus monkeys. We conclude that two nonconsecutive basic residues within the F protein cleavage site are sufficient for efficient replication of human PIV3 in primates.     

Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function.

Eighteen neutralizing monoclonal antibodies (MAbs) specific for the fusion glycoprotein of the A2 strain of respiratory syncytial virus (RSV) were used to construct a detailed topological and operational map of epitopes involved in neutralization and fusion. Competitive binding assays identified three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB). Thirteen MAb-resistant mutants (MARMs) were selected, and the neutralization patterns of the MAbs with either MARMs or RSV clinical strains identified a minimum of 16 epitopes. MARMs selected with antibodies to six of the site A and AB epitopes displayed a small-plaque phenotype, which is consistent with an alteration in a biologically active region of the F molecule. Analysis of MARMs also indicated that these neutralization epitopes occupy topographically distinct but conformationally interdependent regions with unique biological and immunological properties. Antigenic variation in F epitopes was examined by using 23 clinical isolates (18 subgroup A and 5 subgroup B) in cross-neutralization assays with the 18 anti-F MAbs. This analysis identified constant, variable, and hypervariable regions on the molecule and indicated that antigenic variation in the neutralization epitopes of the RSV F glycoprotein is the result of a noncumulative genetic heterogeneity. Of the 16 eptiopes, 8 were conserved on all or all but 1 of 23 subgroup A or subgroup B clinical isolates.     

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.     

Type-specific epitopes targeted by monoclonal antibodies with exceptionally potent neutralizing activities for selected strains of human immunodeficiency virus type 1 map to a common region of the V2 domain of gp120 and differ only at single positions from the clade B consensus sequence

Only a few monoclonal antibodies (MAbs) have been isolated that recognize conserved sites in human immunodeficiency virus type 1 (HIV-1) Env proteins and possess broad neutralizing activities. Other MAbs directed against targets in various domains of Env have been described that are strongly neutralizing, but they possess limited breadth. One such MAb, 2909, possesses a uniquely potent neutralizing activity specific for a quaternary epitope on SF162 Env that requires the presence of both the V2 and the V3 domains. We now show that replacement of the SF162 V3 sequence with consensus V3 sequences of multiple subtypes led to attenuated but still potent neutralization by 2909 and that the main determinants for the type specificity of 2909 reside in the V2 domain. A substitution at position 160 completely eliminated 2909 reactivity, and mutations at position 167 either attenuated or potentiated neutralization by this antibody. Different substitutions at the same positions in V2 were previously shown to introduce epitopes recognized by MAbs 10/76b and C108g and to allow potent neutralization by these MAbs. Two substitutions at key positions in the V2 domain of JR-FL Env also allowed potent expression of the 2909 epitope, and single substitutions in YU2 V2 were sufficient for expression of the 2909, C108g, and 10/76b epitopes. These results demonstrate that the minimal epitopes for 2909, C108g, and 10/76b differed from that of the clade B consensus sequence only at single positions and suggest that all three MAbs recognize distinct variants of a relatively conserved sequence in V2 that is a particularly sensitive mediator of HIV-1 neutralization.     

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades

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

Analysis of bovine herpesvirus 1 glycoprotein gIV truncations and deletions expressed by recombinant vaccinia viruses.

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.     

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.     

Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein.

The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quaternary interactions, we used a panel of 38 conformation-dependent and 9 conformation-independent anti-gp41 monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric Env protein. By cross-competition experiments using these MAbs and several others previously described, six distinct antigenic determinants were identified and mapped. Three of these determinants are conformational in nature and dependent in part on Env oligomeric structure. MAbs to two of these determinants were broadly cross-reactive with Env proteins derived from primary virus strains. The prevalence of antibodies in HIV-1-positive human sera to the antigenic determinants was determined by the ability of such sera to block binding of MAbs to Env protein. Strong blocking activity that correlated with cross-reactivity was found.     

猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析

为了比较猪瘟病毒 (HCV)野毒株、疫苗株及标准株之间E2基因抗原区域的差异 ,采用RT PCR扩增了HCV石门株、兔化弱毒疫苗株、野毒 0 3及 0 7株的囊膜糖蛋白E2 (gp55)全基因的cDNA片段 ,分别克隆于pGEM T载体中并对其进行了核苷酸序列测定及氨基酸序列的推导 ,同时进行了同源性比较及E2结构与功能的分析。所测 4株HCVE2基因的长度均为1 2 73bp,所编码的氨基酸序列均包括部分信号肽序列和完整的跨膜区序列 ,共由 381个氨基酸组成 ;4个毒株E2蛋白N末端的 683位至 690位信号肽序列 (WLLLVTGA)和C末端 1 0 30～1 0 63位跨膜区均为保守序列 ,而且具疏水性 ;N末端抗原功能区中 ,4个E2蛋白与其它所比较序列在位于第 753位至 759位氨基酸处 ,均有一段保守序列RYLASLH ,无一氨基酸发生变异 ,为亲水性 ,在整个E2蛋白抗原谱中抗原性峰值为最高 ,推测对抗原性产生起重要作用 ;4个E2蛋白的氨基酸序列中均含有 1 5个半胱氨酸 (Cys)残基 ,其数量及位置与国外五株HCV(Brescia ,C ,Alfort.ALD和GPE)完全一致。表明…     

Location, exposure, and conservation of neutralizing and nonneutralizing epitopes on human immunodeficiency virus type 2 SU glycoprotein.

Eleven rat monoclonal antibodies (MAbs) that recognize the SU glycoprotein of human immunodeficiency virus type 2 (HIV-2) ROD were produced and characterized. Binding sites for eight of these MAbs were mapped to epitopes within the Cl, V1/V2, C2, and V3 envelope regions. The three other MAbs defined at least two conformation-dependent, strain-specific epitopes outside Vl/V2, V3, and the CD4-binding site. The MAbs were used to probe the tertiary structure of oligomeric envelope glycoprotein expressed on the surfaces of infected cells. Epitopes at the apices of V2 and V3 were exposed on the native molecule, whereas other epitopes on V1/V2, Cl, and C2 were hidden. The MAbs defined three neutralization targets on exposed domains: two linear epitopes in the V2 and the V3 loops and one conformational epitope outside V1, V2, and V3.     

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.     

Identification of domains on the fusion (F) protein trimer that influence the hemagglutinin-neuraminidase specificity of the f protein in mediating cell-cell fusion

For most paramyxoviruses, virus type-specific interaction between fusion (F) protein and attachment protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoprotein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein. In the present study, we further investigated the sites of the F protein that could be critical for determining the HN protein specificity. By analyzing the reported structure of the F protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN protein, suggesting that these domains are involved in determining the HN protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.     

Exploration of antigenic variation in gp120 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies.

The reactivities of a panel of 14 monoclonal antibodies (MAbs) with monomeric gp120 derived from 67 isolates of human immunodeficiency virus type 1 of clades A through F were assessed by using an antigen-capture enzyme-linked immunosorbent assay. The MAbs used were all raised against gp120 or gp120 peptides from clade B viruses and were directed at a range of epitopes relevant to human immunodeficiency virus type 1 neutralization: the V2 and V3 loops, discontinuous epitopes overlapping the CD4-binding site, and two other discontinuous epitopes. Four of the five V3 MAbs showed modest cross-reactivity within clade B but very limited reactivity with gp120s from other clades. These reactivity patterns are consistent with the known primary sequence requirements for the binding of these MAbs. One V3 human MAb (19b), however, was much more broadly reactive than the others, binding to 19 of 29 clade B and 10 of 12 clade E gp120s. The 19b epitope is confined to the flanks of the V3 loop, and these sequences are relatively conserved in clade B and E viruses. In contrast to the limited reactivity of V3 MAbs, CD4-binding site MAbs were much more broadly reactive across clades, two of these MAbs (205-46-9 and 21h) being virtually pan-reactive across clades A through F. Another human MAb (A-32) to a discontinuous epitope was also pan-reactive. The CD4-binding site is strongly conserved between clades; but when considering the epitopes near the CD4-binding site, clade D gp120 appears to be the most closely related to clade B and clade E appears to be the least related. A tentative rank order for these epitopes is B/D-A/C-E/F. V2 MAbs reacted sporadically within and between clades, and no clear pattern was observable. While results from binding assays do not predict neutralization serotypes, they suggest that there may be antigenic subtypes related, but not identical, to the genetic subtypes.     

Antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants

Antigenic variants of vesicular stomatitis virus (VSV) serotypes New Jersey and Indiana (VSV-NJ, VSV-Ind) were selected by using a panel of monoclonal antibodies (MAb) specific for the major surface glycoprotein (G-protein). The reactivity of antigenic variants with the panel of MAb confirmed observations made by competitive binding assays that four distinct antigenic sites (A-D)NJ on the VSV-NJ G-protein and four partially overlapping sites (A, B1, B2, C)Ind on the VSV-Ind G-protein are involved in virus neutralization. Furthermore, subregions within the A epitopes of both serotypes were detected by variant analysis. The frequency of variation at most epitopes was 1 in 10(5) for VSV-NJ and 1 in 10(6) for VSV-Ind. The A3 and C determinants of VSV-Ind, however, defined by MAb that exhibited overlap in binding to other epitopes, appeared to be relatively invariant. Multiple mutations may be necessary to abolish antibody binding at these sites. Overlap of the C group of anti-VSV-Ind MAb with the A epitopes was assigned to the A2 subregion, because variants selected with A2 MAb show reduced binding of C MAb. Heterogeneous antisera from a primary immune response could detect differences in reactivity between variants at the A epitopes and wild-type VSV-NJ or VSV-Ind, suggesting the A epitope is immunodominant. Hyperimmune sera could detect a small difference between ANJ and BNJ variants compared to wild-type VSV-NJ, but could not distinguish between VSV-Ind variants and wild-type VSV-Ind.