Characterization and sequence analyses of antibody-selected antigenic variants of herpes simplex virus show a conformationally complex epitope on glycoprotein H.

Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

The receptor-binding and membrane-fusion properties of influenza virus variants selected using anti-haemagglutinin monoclonal antibodies.

A monoclonal antibody raised against X-31 influenza virus reacted with the majority of natural H3N2 viruses isolated between 1968 and 1982. A number of variants of X-31 and of a receptor-binding mutant of X-31 were selected by the antibody during virus replication in eggs and MDCK cells. Antibody-binding assays indicated that the viruses selected were not antigenic variants and analyses using derivatized erythrocytes showed that their receptor-binding properties differed from those of the parent viruses. The amino acid substitutions in the variants were all located in the vicinity of the receptor-binding site and the structural consequences are discussed in relation to the three-dimensional structure of X-31 HA. In addition all of the variants fused membranes at higher pH than wild-type virus indicating that structural modifications in the distal globular region of HA influence the low pH-induced conformational change required for membrane fusion.     

Bioinformatics models for predicting antigenic variants of influenza A/H3N2 virus

MOTIVATION: Continual and accumulated mutations in hemagglutinin (HA) protein of influenza A virus generate novel antigenic strains that cause annual epidemics. RESULTS: We propose a model by incorporating scoring and regression methods to predict antigenic variants. Based on collected sequences of influenza A/H3N2 viruses isolated between 1971 and 2002, our model can be used to accurately predict the antigenic variants in 1999-2004 (agreement rate = 91.67%). Twenty amino acid positions identified in our model contribute significantly to antigenic difference and are potential immunodominant positions.     

Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.

To analyze the pathogenesis of the neurotropic murine coronavirus JHMV, we used monoclonal antibodies to the E2 viral glycoprotein to select antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 were shown to bind to topographically distinct regions of the E2 molecule, and the variants selected with the two antibodies demonstrated very different disease pictures in mice. Variants selected with J.7.2 were, like the parental virus, highly virulent and caused an acute encephalitic illness. By contrast, J.2.2-selected variants predominantly caused a subacute paralytic disease clinically and extensive demyelination histologically. Antigenic differences among the variants and parental virus were readily demonstrable with anti-E2 monoclonal antibodies. However, no differences between the viruses could be shown in binding studies with monoclonal antibodies directed against either E1 or N, the other two JHMV structural proteins. Since only J.2.2 selected demyelinating variants with reduced neurovirulence, it is likely that this monoclonal antibody recognizes a subregion of the E2 molecule that is particularly important in JHMV pathogenesis.     

M protein (M1) of influenza virus: antigenic analysis and intracellular localization with monoclonal antibodies.

A panel of 16 monoclonal antibodies recognizing M protein (M1) of influenza virus was generated. Competition analyses resulted in localization of 14 monoclonal antibodies to three antigenic sites. Three monoclonal antibodies localized to site 1B recognized a peptide synthesized to M1 (residues 220 to 236) with enzyme-linked immunosorbent assay titers equivalent to or greater than that seen with purified M1; therefore, site 1B is located near the C terminus of M1. Sites 2 and 3 localize to the N-terminal half of M1. Antigenic variation of M proteins was seen when the monoclonal antibodies were tested against 14 strains of type A influenza viruses. Several monoclonal antibodies showed specific recognition of A/PR/8/34 and A/USSR/90/77 M proteins and little or no reactivity for all other strains tested. Immunofluorescence analysis with the monoclonal antibodies showed migration of M protein to the nucleus during the replicative cycle and demonstrated association of M protein with actin filaments in the cytoplasm. Use of a vaccinia virus recombinant containing the M-protein gene demonstrated migration of M protein to the nucleus in the absence of synthesis of gene products from other influenza virus RNA segments.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

Humoral immunostimulation. VII. Sialic acid masks antigenic sites on an antibody-selected bariant cell line.

Variant cell lines (LC1, LC2) obtained by growth of mouse L cells (L) in cytostimulatory and cytotoxic doses, respectively, of rabbit anti-L cell antiserum AL) were found previously to be altered in many ways relative to the parent cell line. A major change was the reduction of those surface membrane antigens that AL recognizes. These cell variants have now been found to have increased membrane sialic acid relative to L. Treatment of intact variant cells with neuraminidase (50 units/ml, 37 degrees C, 1 hr) greatly restored the susceptibility of LC1 to lysis with AL. In the presence of 1/100 dilution of AL and 5% complement the viability indices (1.00 = no cell kill) of untreated and neuraminidase-treated cells were respectively: L 0.10 and 0.03 and LC1 0.91 and 0.40. Neuraminidase-treated LC2 cells retained their resistance to AL. Parallel studies with 125I-ALIgG showed increased binding to neuraminidase-treated LC1 relative to native LC1. These findings suggest that the altered membrane sialic acid content affects the immunologic behavior of this cell variant by masking the original cell surface antibody-binding sites. This represents a possible mechanism for tumors to escape immunologic control.     

Characterization of monoclonal antibodies specific for sequential influenza A/PR/8/34 virus variants

A panel of monoclonal antibodies specific for a corresponding panel of sequentially selected variants of influenza A/PR/8/34 virus has been established. Although the monoclonal antibodies are paratypically distinct, idiotypic relatedness has been observed. Two cross-reactive idiotypes have been defined that are associated with the 7183 and S107 VH gene families, respectively. Three of the four monoclonal antibodies utilize the VK21 group of light chains, and three VH genes belong to the VH7183 family and one to the VH S107 family. Antibodies encoded by genes deriving from the VH7183 family share a cross-reactive idiotype, a marker of the VH region as well as distinct individual idiotopes. These antibodies are produced by different clones using related VH and VK genes.     

Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition.

When presented together on the intact influenza virus particle, the external hemagglutinin (HA) and neuraminidase (NA) antigens are competitive, with HA dominant over NA in both T- and B-cell priming (B. E. Johansson, T. M. Moran, and E. D. Kilbourne, Proc. Natl. Acad. Sci. USA 84:6869-6873, 1987). Dissociation and purification of HA and NA from virus and their injection separately or in combination into BALB/c mice eliminates their antigenic competition as measured by antibody response, confirming that it is their structural association that leads to what we have termed intravirionic antigenic competition. We discuss this phenomenon with respect to previously described intermolecular antigenic competition and with regard to its probable mechanism. Our findings are relevant to contemporary interest in viral vaccine vectors and multicomponent vaccines.     

Radioimmunological analysis of the antigenic variability of influenza virus nucleoprotein

Influenza A virus ability to bind anti-NP monoclonal antibodies to two viral strains has been studied by radioimmunoassay on polyethylene film with the subsequent autoradiographic registration of results. Monoclonal antibodies were obtained to the viral strains differing in antigenic formula of outer glycoproteids and isolated at different time. The studied influenza viruses were divided into seven groups due to their ability to bind monoclonal antibodies. The absence of correlation between the antigenic properties of nucleoprotein and glycoproteids has been registered. Variability of some antigenic sites has been analyzed. The human epidemic strains of influenza virus are different in ability to bind monoclonal antibodies from the viral strains that are connected with animals in nature or laboratory practice.     

Restriction endonuclease DNA analysis of antigenic variants of leptospires selected by monoclonal antibodies

The genome of antigenic variant CV (CT3)-1 derived from Leptospira interrogans serovar canicola was compared by cleavage with restriction endonucleases with the parent and serovar bafani, to which the variant was serologically most closely related. No differences were observed between the parent and variant in DNA restriction endonuclease patterns using eight restriction endonucleases. Serovar bafani was different in the patterns from the parent and antigenic variant CV (CT3)-1. The two antigenic variants derived from serovar hebdomadis, HV (H16)-1 and HV (H19)-1 which belonged serologically to serovars jules and hebdomadis, respectively, were compared by restriction endonuclease DNA analysis with the parent and serovar jules. No differences were observed between the parent and variants in DNA restriction endonuclease patterns using the same enzymes. But some differences were observed in DNA restriction endonuclease patterns between HV (H16)-1 and serovar jules. Thus, the antigenic variant selected from the parent by the anti-parent monoclonal antibody and serologically different from the parent, being identified either as a new serovar or as a known one, was found to be similar to the parent by the restriction endonuclease DNA analysis.     

Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies.

Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.     

Comparison of epitope structures of H3HAs through protein modeling of influenza A virus hemagglutinin: mechanism for selection of antigenic variants in the presence of a monoclonal antibody

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site-directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding-positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three-dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding-positive mutant seemed to be explained by the changes noted on this model.     

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.     

Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. We found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of the three epitopes are located in nonoverlapping domains on the matrix protein molecule.