Antigenic variation (mar mutations) in herpes simplex virus glycoprotein B can induce temperature-dependent alterations in gB processing and virus production.

Monoclonal antibody-resistant (mar) mutants altered in the antigenic structure of glycoprotein B (gB) of herpes simplex virus type 1, strain KOS-321, were selected by neutralization with each of six independently derived gB-specific monoclonal antibodies. Analysis of the reactivity patterns of these mar mutants with a panel of 16 virus-neutralizing monoclonal antibodies identified at least five nonoverlapping epitopes on this antigen, designated groups I through V. Multiple mar mutations were also introduced into the gB structural gene by recombination and sequential antibody selection to produce a set of mar mutants with double, triple, and quadruple epitope alterations. Group II (B2) and group III (B4) antibodies were used to select the corresponding mutants, mar B2.1 and mar B4.1, which in addition to carrying the mar phenotype were temperature sensitive (ts) for processing of the major partially glycosylated precursor of gB, pgB (Mr = 107,000), to mature gB (Mr = 126,000) and showed reduced levels of gB on the cell surface at high temperature (39 degrees C). These mutants were not, however, ts for production of infectious progeny. A recombinant virus, mar B2/4.1, carrying both of these alterations was ts for virus production and failed to produce and transport any detectable mature gB to the cell surface at 39 degrees C. Rather, pgB accumulated in the infected cell. Revertants of the ts phenotype, isolated from virus plaques at 39 degrees C, regained the B2 but not the B4 epitope and were phenotypically indistinguishable from the mar B4.1 parent. Finally, it was shown that group II (B5) and group III (B4) antibodies failed to immunoprecipitate pgB (39 degrees C) produced by ts gB mutants of herpes simplex virus type 1 which were not selected with monoclonal antibodies. Taken together, our findings indicate that (i) mar mutations can alter antigenic as well as other functional domains of gB, namely, the domain(s) involved in processing and infectivity, and (ii) group II and group III epitopes lie within an essential functional domain of gB which is a target for ts gB mutations.     

Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus penetration.

Analysis of six monoclonal antibody-resistant (mar) mutants in herpes simplex virus type 1 glycoprotein B identified two type-common (II and III) and two type-specific (I and IV) antigenic sites on this molecule. To derive additional information on the location of these sites, mar mutations were mapped and nucleotide alterations were identified by DNA sequencing. Each mutant carried a single amino acid substitution resulting from a G-to-A base transition. Alterations affecting antibody neutralization were identified at residues 473, 594, 305, and 85 for mutants in sites I through IV, respectively. Two clonally distinct site II antibodies each selected mar mutants (Gly to Arg at residue 594) that exhibited a reduction in the rate of entry (roe) into host cells. A site II mar revertant that regained sensitivity to neutralization by site II antibodies also showed normal entry kinetics. DNA sequencing of this virus identified a single base reversion of the site II mar mutation, resulting in restoration of the wild-type sequence (Arg to Gly). This finding demonstrated that the mar and roe phenotypes were the result of a single mutation. To further define structures that contributed to antibody recognition, monoclonal antibodies specific for all four sites were tested for their ability to immune precipitate a panel of linker-insertion mutant glycoprotein B molecules. Individual polypeptides that contained single insertions of 2 to 28 amino acids throughout the external domain were not recognized or were recognized poorly by antibodies specific for sites II and III, whereas no insertion affected antibody recognition of sites I and IV. mar mutations affecting either site II or III were previously shown to cause temperature-sensitive defects in glycoprotein B glycosylation, and variants altered in both these sites were temperature sensitive for virus production. Taken together, the data indicate that antigenic sites II and III are composed of higher-order structures whose integrity is linked with the ability of glycoprotein B to function in virus infectivity.     

Heterogeneity of the mutation rates of influenza A viruses: isolation of mutator mutants.

The rates of mutation to the mar (monoclonal antibody-resistant) genotype of individual influenza virus plaque isolates, obtained from a stock generated after two successive cloning steps, have been determined by the fluctuation test. When a random sample of 60 clones was analyzed, 7 contained a proportion of mar mutants significantly higher than the average, and among them, 2 showed a mutation rate two to three times higher than the average value obtained for the virus population when the hemagglutinin-specific monoclonal antibody 2G10 was used. In order to look for mutants with higher mutation rates, a systematic search was carried out with a nonmutagenized virus stock, and several clones with increased mutation rates were isolated. One of them (mut43) was characterized further and was shown to have a mutation rate three to four times higher than that of the virus population at the sites defined by two nonoverlapping, hemagglutinin-specific monoclonal antibodies as well as at the site defined by a neuraminidase-specific monoclonal antibody. These results indicate that the mutation rate of an influenza virus is a weighted average of the contributions of a heterogeneous population. The consequences of this fact for the adaptive evolution of influenza viruses are discussed.     

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.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Epitopes of herpes simplex virus type 1 glycoprotein gC are clustered in two distinct antigenic sites.

Epitopes of herpes simplex virus type 1 (HSV-1) strain KOS glycoprotein gC were identified by using a panel of gC-specific, virus-neutralizing monoclonal antibodies and a series of antigenic variants selected for resistance to neutralization with individual members of the antibody panel. Variants that were resistant to neutralization and expressed an antigenically altered form of gC were designated monoclonal antibody-resistant (mar) mutants. mar mutants were isolated at frequencies of 10(-3) to 10(-5), depending on the antibody used for selection. The epitopes on gC were operationally grouped into antigenic sites by evaluating the patterns of neutralization observed when a panel of 22 antibodies was tested against 22 mar mutants. A minimum of nine epitopes was identified by this process. Three epitopes were assigned to one antigenic site (I), and six were clustered in a second complex site (II) composed of three distinct subsites, IIa, IIb, and IIc. The two antigenic sites were shown to reside in physically distinct domains of the glycoprotein, by radioimmunoprecipitation of truncated forms of gC. These polypeptides lacked portions of the carboxy terminus and ranged in size from approximately one-half that of the wild-type molecule to nearly full size. Antibodies recognizing epitopes in site II immunoprecipitated the entire series of truncated polypeptides and thereby demonstrated that site II resided in the N-terminal half of gC. Antibodies reactive with site I, however, did not immunoprecipitate fragments smaller than at least two-thirds the size of the wild-type polypeptide, suggesting that site I was located in the C-terminal portion. Sites I and II were also shown to be spatially separate on the gC polypeptide by competition enzyme-linked immunosorbent assay with monoclonal antibodies representative of different site I and site II epitopes.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

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.     

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.     

Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50.

A pseudorabies virus variant ( mar197 -1) containing a mutation in a viral glycoprotein with a molecular weight of 50,000 ( gp50 ) was isolated by selecting for resistance to a neurtralizing monoclonal antibody ( MCA50 -1) directed against gp50 . This mutant was completely resistant to neutralization with MCA50 -1 in the presence or absence of complement, and was therefore defined as a mar (monoclonal-antibody-resistant) mutant. The mutation did not affect neutralization with polyvalent immune serum. The mar197 -1 mutant synthesized and processed gp50 normally, but the mutation prevented the binding and immunoprecipitation of gp50 by MCA50 -1. Thus, the mutation was within the structural portion of the gp50 gene affecting the epitope of the monoclonal antibody. The mutation was mapped by marker rescue with cloned pseudorabies restriction enzyme fragments to the short region of the pseudorabies genome between 0.813 and 0.832 map units. This is equivalent to a 2.1-kilobase-pair region.     

Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus.

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.     

Neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein.

Neuroattenuated variants of mouse hepatitis virus type 4 (MHV-4) selected for resistance to neutralizing monoclonal antibodies (R.G. Dalziel, P.W. Lampert, P. J. Talbot, and M. J. Buchmeier, J. Virol. 59:463-471, 1986) were found to harbor large deletions in both mRNA 3 and its protein product, the 180-kilodalton viron spike (S) glycoprotein. By using antipeptide antibodies directed against selected portions of the chain, deletions were mapped to the middle of the amino-terminal S1 fragment, one of the two posttranslational cleavage products of S, and involved omission of 15 kilodaltons of protein. Deletion mutants could be selected only after multiple passage of virus through cultured cell lines; minimally passaged MHV-4 stocks contained putative point mutants selectable by neutralizing monoclonal antibodies but no deletions. Enhanced growth of deletion mutants relative to wild-type virus was observed in four cell lines used for virus propagation and was attributed to delayed and diminished cytopathic effects that allowed cultures to support virus production for prolonged periods. This hypothesis was reinforced by the finding that no selective advantage for the deletion mutants was observed in two cell lines resistant to virus-induced cytopathic effects. These results indicate that the passaging of MHV-4 in culture generates heterogeneity in S structure and eventually selects for rare neutralization-resistant deletion mutants with decreased virulence properties.     

The neuraminidase of influenza virus

It is the enzyme neuraminidase, projecting from the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3-dimensional structure of the epitopes was obtained by X-ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.     

Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach.     

Genetics of eye-gap and maroon-eye mutants in the mosquito.

Linkage analysis of two new eye mutants, eye-gap (e) and maroon-eye (mar), in the mosquito Culex pipiens, is presented. Both mutants are sex-linked, recessive, and demonstrate complete penetrance and high expressivity. The gene order is e-m-mar. The most representative map distances in e-m and m-mar segments are 17.7 and 26.1, respectively. Our maroon-eye was isolated from Belem, Brazil and is allelic to another maroon-eye mutant isolated from Lake Charles, Louisiana. However, while mar (Lake Charles) straddles along the sex locus, m (map distance approximately 1 unit), the mar (Belem) gene is far removed from m (map distance 26.1 units).     

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).     

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase.

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.     

Demonstration of an immunodominant neutralization site by analysis of antigenic variants of SA11 rotavirus

Serotype-specific monoclonal antibodies were used to select mutants of SA11 rotavirus that were resistant to neutralization. The antigenic characteristics of these mutants were studied with with a panel of monoclonal antibodies. We isolated one type of mutant which showed a dramatic increase (greater than 10-fold) in resistance to neutralization by hyperimmune antiserum, and this together with other data indicates the presence on the rotavirus major outer shell glycoprotein of an immunodominant antigenic site involved in virus neutralization. The mutants were also useful in classifying neutralizing monoclonal antibodies.     

High mutant frequency in populations of a DNA virus allows evasion from antibody therapy in an immunodeficient host

The degree of genetic heterogeneity of DNA virus populations in nature and its consequences for disease control are virtually unknown. The parvovirus minute virus of mice (MVMi) was used here to investigate (i) the frequency of antibody-escape mutants in populations of a DNA virus and (ii) the ability of a DNA virus to evade in the long-term a passive monoclonal antibody (MAb) therapy in an immunodeficient natural host. Independent clonal populations of MVMi harbored a high proportion of mutants resistant to neutralizing MAb (mutant frequency = [2.8 +/- 0.5] x 10(-5)) that rapidly evolved under antibody pressure in culture to become mixtures dominated by genotypically diverse escape mutants. Immunodeficient mice naturally infected with clonal populations of MVMi and subsequently treated by intravenous injections of MAb were initially protected from the characteristic viral induced lethal leukopenia. However, some treated animals developed a delayed severe leukopenic syndrome associated with the emergence of genetically heterogeneous populations of MAb-resistant mutants in the MVMi main target organs. The 11 plaque-purified viruses analyzed from an antibody-resistant population obtained from one animal corresponded to four different mutant genotypes, although their consensus sequence remained wild type. All cloned escape mutants harbored single radical amino acid changes within a stretch of seven residues in a surface-exposed loop at the threefold axes of the capsid. This antigenic site, which can tolerate radical changes preserving MVMi pathogenic potential, may thereby allow the virus to evade the immune control. These findings indicate a high genetic heterogeneity and rapid adaptation of populations of a mammal DNA virus in vivo and provide a genetic basis for the failure of passive immunotherapy in the natural host.