Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of a monoclonal antibody specific to Escherichia coli H33 flagellin by using predetermined polypeptides

The Escherichia coli H serogroup is determined by flagellin, which has both H-type-specific and cross-reactive epitopes. The cross-reactive epitopes are responsible for the cross-reaction found in agglutination. To identify the specific epitope in H33 flagellin, the H33 flagellin gene was sequenced and the encoded central variable region (CVR) was determined. Four overlapping fragments of the CVR were prepared and their specificity was verified using different H-type antisera. Short fragments carrying potential H-type-specific determinants were selected, and monoclonal antibodies (MAbs) against these fragments were prepared. A murine MAb of subtype IgG1 showing specificity to H33 flagellin was produced. The epitope of the MAb was mapped to amino acid residues 250-260.     

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.     

Common Neutralization Epitope in Minor Capsid Protein L2 of Human Papillomavirus Types 16 and 6

Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.     

Identification of Immunodominant Neutralizing Epitopes on the Hemagglutinin Protein of Rinderpest Virus

The immunodominant epitopes on the hemagglutinin protein of rinderpest virus (RPV-H) were determined by analyzing selected monoclonal antibody (MAb)-resistant mutants and estimating the level of antibody against each epitope in five RPV-infected rabbits with the competitive enzyme-linked immunosorbent assay (c-ELISA). Six neutralizing epitopes were identified, at residues 474 (epitope A), 243 (B), 548 to 551 (D), 587 to 592 (E), 310 to 313 (G), and 383 to 387 (H), from the data on the amino acid substitutions of hemagglutinin protein of MAb-resistant mutants and the reactivities of MAbs against RPV-H to the other morbilliviruses. The epitopes identified in this study are all positioned on the loop of the propeller-like structure in a hypothetical three-dimensional model of RPV-H (J. P. M. Langedijk et al., J. Virol. 71:6155-6167, 1997). Polyclonal sera obtained from five rabbits infected experimentally with RPV were examined by c-ELISA using a biotinylated MAb against each epitope as a competitor. Although these rabbit sera hardly blocked binding of each MAb to epitopes A and B, they moderately blocked binding of each MAb to epitopes G and D and strongly blocked binding of each MAb to epitopes E and H. These results suggest that epitopes at residues 383 to 387 and 587 to 592 may be immunodominant in humoral immunity to RPV infection.     

Localization of monoclonal antibody epitopes and functional domains in the hemagglutinin protein of measles virus.

The expression of chimeric proteins was performed for the localization of monoclonal antibody (MAb) epitopes and functional domains in the hemagglutinin (H) protein of measles virus. The fusion helper function of the H protein was ablated by a single amino acid substitution at residue 98. Loss of reactivity to MAb 79-XV-V17 and to MAbs 16-CD-11 and 80-II-B2 was attributed to substitutions between residues 211 and 291 and between 451 and 505, respectively. The 80-II-B2 MAb epitope also seemed to be within a domain required for hemadsorption and hemagglutination activities.     

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.     

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.     

Nonbactericidal antibodies against Neisseria gonorrhoeae: evaluation of their blocking effect on bactericidal antibodies directed against outer membrane antigens

Nonbactericidal monoclonal antibodies (MAbs) directed against gonococcal surface antigens were examined for their effect on complement-mediated bactericidal killing by other MAbs and normal human serum. One MAb, SM73, directed against the H.8 antigen activated complement only moderately well and had little influence on bactericidal antibodies. Two antibodies directed against an epitope on protein III had very different effects. Antibody SM51 activated complement poorly and had no effect on bactericidal killing, whereas antibody SM50, although itself nonbactericidal, activated complement and blocked the bactericidal effect of other antibodies. The extent of the blocking ability of MAb SM50 was studied using MAbs of different specificities as well as polyclonal antisera raised against gonococcal surface antigens. Antibody SM50 blocked IgG MAbs of all specificities, but several MAbs of the IgM class retained their bactericidal effect. Each of these IgM MAbs reacted with lipopolysaccharide, but with different epitopes.     

Interspecies cross-reactivity of monoclonal antibodies to various epitopes of human plasminogen

The immunological cross-reactivities of three conformationally specific monoclonal antibodies to distinct epitopes on human plasminogen toward plasminogens purified from 14 additional species have been examined. Antibody 10-F-1, which is produced against an epitope on the kringle 4 region of human plasminogen, shows a high degree (greater than 80%) of cross-reactivity against baboon, goat, monkey, ovine, and rabbit plasminogens; more limited (20-50%) cross-reactivity against bovine, equine, goose, guinea pig, mouse, rat, and porcine plasminogens; and little comparable cross-reactivity against canine and chicken plasminogens. Antibody 10-H-2, generated to an epitope of the kringles 1-3 region of human plasminogen, shows extensive cross-reactivity (72%) only toward monkey plasminogen, more limited (22-35%) cross-reactivity toward equine and rabbit plasminogens, and much less cross-reactivity toward any other of the above plasminogens. Antibody 10-V-1, also produced against an epitope on the kringle 1-3 region of human plasminogen, which is distinct from the 10-H-2 epitope, shows extensive cross-reactivity (72-100%) with baboon, monkey, and rabbit plasminogens; more limited cross-reactivity with equine (48%) and mouse (28%) plasminogens; and a low level of such reactivity with the remaining plasminogens. These studies show that the extent of interspecies cross-reactivity of various plasminogens greatly depends upon the epitope in question. The K4 region of these molecules appears more extensively conserved than the K1-3 region, at least in regard to the particular epitopes examined in this study.     

Fine mapping of antigenic site II of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) which plays an important role in viral infection and pathogenesis. Previously, anti-gD monoclonal antibodies (MAbs) were arranged into groups which recognize distinct type-common and type-specific sites on HSV-1 gD (gD-1) and HSV-2 gD (gD-2). Several groups recognize discontinuous epitopes which are dependent on tertiary structure. Three groups, VII, II, and V, recognize continuous epitopes present in both native and denatured gD. Previously, group II consisted of a single MAb, DL6, whose epitope was localized between amino acids 268 and 287. In the study reported here, we extended our analysis of the antigenic structure of gD, concentrating on continuous epitopes. The DL6 epitope was localized with greater precision to residues 272 to 279. Four additional MAbs including BD78 were identified, each of which recognizes an epitope within residues 264 to 275. BD78 and DL6 blocked each other in binding to gD. In addition, a mutant form of gD was constructed in which the proline at 273 was replaced by serine. This change removes a predicted beta turn in gD. Neither antibody reacted with this mutant, indicating that the BD78 and DL6 epitopes overlap and constitute an antigenic site (site II) within residues 264 to 279. A separate antigenic site (site XI) was recognized by MAb BD66 (residues 284 to 301). This site was only six amino acids downstream of site II, but was distinct as demonstrated by blocking studies. Synthetic peptides mimicking these and other regions of gD were screened with polyclonal antisera to native gD-1 or gD-2. The results indicate that sites II, V, VII, and XI, as well as the carboxy terminus, are the major continuous antigenic determinants on gD. In addition, the results show that the region from residues 264 through 369, except the transmembrane anchor, contains a series of continuous epitopes.     

Heterogeneity of apolipoprotein E epitope expression on human lipoproteins: importance for apolipoprotein E function

The conformations of apolipoproteins on the surfaces of lipoprotein particles affect their physiologic functions. The conformations of apoE on plasma lipoproteins were examined using a panel of eight anti-apoE monoclonal antibodies (MAbs). The antibodies, which reacted with the major isoforms of apoE (E2, E3, and E4), defined at least five epitopes on apoE. Proteolytic fragments and synthetic peptides of apoE were used in binding assays to assign antibody epitopes; the epitopes were all localized to the middle third of the apoE molecule. The expression of apoE epitopes on isolated apoE and on lipoproteins was probed in competitive microtiter plate immunoassays using the anti-apoE MAbs, 125I-labeled apoE as tracer, and isolated apoE, intermediate density (IDL), very low density (VLDL1-3), and high density (HDL2 and HDL3) lipoproteins as competitors. The antibodies determined the patterns of competition exhibited by the lipoprotein preparations. Antibodies of the IgM class (WU E-1, WU E-2, WU E-3) defined two sets of conformation-dependent epitopes that were assigned towards the middle and the carboxyl terminal of the middle third of apoE. Competition curves using these antibodies, apoE, and lipoproteins showed a large variability in ED50 values. MAbs WU E-4, WU E-7, and WU E-10 defined epitopes near the receptor recognition site on apoE. Competition curves demonstrated small ranges of ED50 values. MAbs WU E-11 and WU E-12, which defined epitopes toward the amino-terminal region of apoE, exhibited competition curves for apoE and lipoproteins that had consistent, but wider ranges of ED50 values. There was no strict relationship between lipoprotein flotation rates and epitope expression for any of the MAbs. Immunoaffinity chromatography of VLDL subfractions on four different MAb columns indicated that the differences in the competitive abilities of VLDL subfractions were partly due to heterogeneity of apoE epitope expression within any population of particles. VLDL particles specifically retained on two different anti-apoE MAb columns were better competitors than unretained fractions for 125I-labeled LDL binding to the apoB, E-receptor of cultured human fibroblasts, suggesting that increased accessibility of apoE on the surface of VLDL is associated with increased receptor recognition. These data suggest that individual epitopes of apoE can be modulated; epitope expressions are not determined solely by the sizes and/or densities of lipoprotein particles; and differences in apoE conformation have significant metabolic consequences.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.     

Antigenic analysis of potato virus A particles and coat protein

Five monoclonal antibodies (MAbs) were prepared to particles of potato virus A (PVA), isolate B11. In immunoblots, MAbs A1D8 and A5B6 reacted only with full length molecules of PVA coat protein (CP). Pepscan tests with overlapping octapeptides representing the whole sequence of PVA CP showed that the epitope detected by MAb A5B6 is contained in its N-terminal octapeptide. MAbs A9A4, A3H4 and A6B8 reacted with CP molecules that lacked about 5 kD of sequence at their end(s) and detected epitopes at residues 52 to 62, 64 to 73 and 75 to 82 respectively, all of which lie in the protease-resistant core of the CP. The epitope which reacts with MAb A3H4 is in a region predicted to be hydrophobic and is not detected in intact virus particles, indicating it is a cryptotope. In contrast, MAbs A6B8 and A9A4 reacted with freshly purified PVA particles but more strongly with partially degraded ones. Pepscan tests with polyclonal antibodies to PVA isolate B11 identified five additional immunogenic sequences in PVA CP and showed that regions at the N-termini of the intact and core molecules are immunodominant. PVA isolate B11 was not transmitted by aphids, and its CP N-terminal octapeptide contains the sequence DAS, which is associated with aphid-non-transmissibility in other potyviruses. MAb A5B6, which detects this region, reacted strongly in ELISA with three out of four other aphid-non-transmissible PVA isolates but only weakly with three aphid-transmissible ones, suggesting that differences in N-terminal sequence may underlie most of the differences in aphid transmissibility.     

Molecular location of a species-specific epitope on the hamster scrapie agent protein.

Scrapie is a transmissible neurodegenerative disease of sheep and goats. An abnormal host protein, Sp33-37, is the major protein component of the scrapie agent and the only known disease- or agent-specific macromolecule. Two monoclonal antibodies (MAbs), 4H8 (immunoglobulin G2b [IgG2b]) and 6B11 (IgG1), produced by immunizing mice with the intact hamster 263K scrapie agent protein, Sp33-37Ha, were found to have species specificity similar to that reported previously for MAb 3F4 (IgG2a), which was produced by using PrP-27-30 as the immunogen (R. J. Kascsak, R. Rubenstein, P. A. Merz, M. Tonna-DeMasi, R. Fersko, R. I. Carp, H. M. Wisniewski, and H. Diringer, J. Virol. 61:3688-3693, 1987). These antibodies all bound to Sp33-37 derived from hamster but not from mouse cells. Competitive binding assays demonstrated that all three MAbs bound to the same or overlapping sites on Sp33-37Ha. The molecular location of the epitope for these antibodies was determined to within 10 residues by using an antigen competition enzyme-linked immunosorbent assay in which synthetic peptides spanning Sp33-37Ha residues 79 to 93 or 84 to 93 specifically inhibited binding of these antibodies to plates coated with purified Sp33-37Ha. A synthetic peptide with the mouse-specific sequence (83 to 92) that differed from the hamster sequence by substitution at two positions (MetHa-87----LeuMo-86 and MetHa-90----ValMo-89) did not inhibit antibody binding to Sp33-37Ha. MAb 3F4 binding to hamster Sp33-37 was eliminated by chemical modification of Sp33-37Ha with diethylpyrocarbonate or succinic anhydride and by cleavage with CNBr or trypsin. The effect of diethylpyrocarbonate on MAb 3F4 binding was not reversed by hydroxylamine treatment. MAb 3F4 binding was not affected by prolonged exposure of Sp33-37Ha to 70% formic acid or by boiling in sodium dodecyl sulfate. We conclude that the epitope for these MAbs is a linear determinant that includes Met-87, Lys-88, and Met-90 and that Met-90 is probably the major species-specific determinant.     

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.     

Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002

Noroviruses are the primary cause of epidemic gastroenteritis in humans, and GII.4 strains cause ～80% of the overall disease burden. Surrogate neutralization assays using sera and mouse monoclonal antibodies (MAbs) suggest that antigenic variation maintains GII.4 persistence in the face of herd immunity, as the emergence of new pandemic strains is accompanied by newly evolved neutralization epitopes. To potentially identify specific blockade epitopes that are likely neutralizing and evolving between pandemic strains, mice were hyperimmunized with GII.4-2002 virus-like particles (VLPs) and the resulting MAbs were characterized by biochemical and immunologic assays. All of the MAbs but one recognized GII.4 VLPs representing strains circulating from 1987 to 2009. One MAb weakly recognized GII.4-1987 and -1997 while strongly interacting with 2002 VLPs. This antibody was highly selective and effective at blocking only GII.4-2002-ligand binding. Using bioinformatic analyses, we predicted an evolving GII.4 surface epitope composed of amino acids 407, 412, and 413 and subsequently built mutant VLPs to test the impact of the epitope on MAb binding and blockade potential. Replacement of the 2002 epitope with the epitopes found in 1987 or 2006 strains either reduced or ablated enzyme immunoassay recognition by the GII.4-2002-specific blockade MAb. These data identify a novel, evolving blockade epitope that may be associated with protective immunity, providing further support for the hypotheses that GII.4 norovirus evolution results in antigenic variation that allows the virus to escape from protective herd immunity, resulting in new epidemic strains.     

A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.