Monoclonal antibodies to gonococcal outer membrane protein IB: use in investigation of the potential protective effect of antibodies directed against conserved and type-specific epitopes

Several monoclonal antibodies directed against gonococcal outer membrane protein IB have been used in in vitro assays to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, virulence of the variant P9-17 for epithelial cells in tissue culture was reduced in the presence of three of the four antibodies which recognized type-specific epitopes. Similarly, virulence of P9-17 as well as a recent isolate was reduced in the presence of the one antibody, SM24, which reacted with a conserved epitope. This antibody was also bactericidal in the presence of complement, and in addition was opsonic for several protein IB-expressing strains as determined by polymorphonuclear leucocyte chemiluminescence measurements. Similarly, all the type-specific antibodies were opsonic for P9 variants. However, only two of these antibodies mediated complement-dependent killing although those which were ineffective were nevertheless complement-fixing antibodies. These results indicate that antibodies to closely positioned epitopes on protein I vary in their biological activities and that the conserved epitope recognized by the antibody SM24 is potentially an effective target on the gonococcal surface for immunoprophylaxis.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Immunobiology of gonococcal outer membrane protein I

Monoclonal antibodies have been obtained which react with gonococcal outer membrane protein I. One antibody recognised the majority of strains expressing P.IA and another recognised the majority of strains expressing P.IB. In in vitro tests both antibodies were bactericidal in the presence of complement, opsonic for phagocytosis by human PMN and protected epithelial cells against gonococcal invasion. Thus conserved epitopes on P.I. are potentially effective targets for immunoprophylaxis.     

The potential protective effect of monoclonal antibodies to gonococcal outer membrane protein IA

Hybrid cell lines were derived which produced monoclonal antibodies directed against gonococcal outer membrane protein IA. One antibody, SM101, recognized 24 P.IA-expressing strains out of 25 tested--the rest exhibited relatively less cross-reactivity. Competitive radioimmunoassays revealed that each antibody could effectively inhibit binding of the others, suggesting close proximity of the epitopes recognized. The antibodies were used in assays in vitro to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, the antibodies afforded some protection to epithelial cells challenged with gonococci. They were very effective at bactericidal killing in the presence of complement and, in addition, were opsonic for homologous and heterologous strains. The cross-reacting antibody, SM101, was one of the most effective in both assays. The results show that the conserved epitope on P.IA recognized by antibody SM101 is potentially an effective target on the gonococcal surface for immunoprophylaxis.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

The nucleolar RNA-binding protein B-36 is highly conserved among plants

The nucleolar protein B-36 is an RNA-associated protein which has a number of properties in common with pre-mRNA-binding proteins (hnRNP proteins). Like the hnRNP proteins, B-36 appears to be evolutionarily conserved among various eukaryotes (protists and several animal species). The conservation of B-36 throughout the plant kingdom has been investigated using a panel of nine monoclonal antibodies previously shown to recognize a minimum of four different epitopes in Physarum B-36, the protein used to generate the monoclonal antibodies. Two of the epitopes (I and III) are widely conserved in 34 kDa proteins (presumably B-36 homologues) from the various species tested (Chlamydomonas, moss, fern, oat, onion, carrot, and bean). Using immunofluorescence localization in moss and carrot protoplasts, the cross-reacting proteins were shown to be restricted to the nucleolus, further confirming their probable homology to B-36. Epitopes I and III are also unique to the B-36 homologues as demonstrated by the failure of any other bands to cross-react. Another epitope (IV) was specifically recognized in the plant B-36 homologues but exhibited greatly reduced affinity for the monoclonal antibody relative to Physarum B-36. The remaining epitope (II), unlike the others, exhibited variable conservation in the plant B-36 homologues and, in addition, was present in several other seemingly unrelated proteins. Finally, several of the plant species exhibited two cross-reacting variants at roughly the 34 kDa position and in at least one of these cases a single monoclonal antibody was able to distinguish between the two variants, a result indicating that the variants do have bona fide structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种蛋白质B细胞表位展示系统的构建及验证

目的:建立一种能够有效展示蛋白质B细胞表位的载体系统,以用于表位特异性抗体的制备和表位疫苗的设计。方法:选用抗体的可变区作为蛋白质B细胞表位展示的骨架蛋白,B细胞表位的展示部位为CDR3区。全基因合成骨架蛋白基因,通过重叠PCR将B细胞表位编码序列插入到骨架蛋白基因中,原核表达目的蛋白,利用Sephacryl S-100层析柱进行蛋白纯化,用纯化的蛋白按常规方法免疫小鼠,采用ELISA法检测免疫血清的滴度及特异性,通过Western blot和间接免疫荧光技术进一步验证免疫血清对目的蛋白的识别。结果:成功构建了3种表位展示蛋白,均表现出了很好的抗原性,表位展示蛋白免疫血清具有较好的特异性,能够特异识别所展示的B细胞表位。结论:抗体可变区作为骨架蛋白能够很好地展示B细胞表位,免疫小鼠后获得的免疫血清表现出了较好的特异性。     

Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection.     

Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases

gp120 is a subunit of the envelope glycoprotein of HIV-1. The third variable loop region of gp120 (V3 loop) contains multiple immunodominant epitopes and is also functionally important for deciding cell-tropism of the virus. 447-52D is a monoclonal antibody that recognizes the conserved tip of the V3 loop in a beta-turn conformation. This antibody has previously been shown to neutralize diverse strains of the virus. In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, the known epitope of 447-52D was inserted at three different surface loop locations in the small, stable protein Escherichia coli Trx (thioredoxin). At one of the three locations (between residues 74 and 75), the insertion was tolerated, the resulting protein was stable and soluble, and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, the V3 peptide-inserted Trx scaffold was able to generate anti-V3 antibodies that could compete out 447-52D binding to gp120. Epitope mapping studies demonstrated that these anti-V3 antibodies recognized the same epitope as 447-52D. Although the 447-52D-type antibodies were estimated to be present at concentrations of 50-400 microg/ml of serum, these were not able to effect neutralization of strains like JRFL and BAL but could neutralize the sensitive MN strain. The data suggest that because of the low accessibility of the V3 loop on primary isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.     

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.     

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.     

Monoclonal antibodies against different epitopes of a 40 Kd capsid protein of infectious bursal disease viruses.

Nine monoclonal antibodies (Mab) against a 40 Kd capsid protein of infectious bursal disease virus (IBDV) strain P3009 were isolated. They were characterized by enzyme-linked immunosorbent assay, indirect fluorescent antibody staining and virus neutralization. They were divided into two groups concerning virus neutralization. Group I Mabs were able to neutralize virus infectivity; however, group II Mabs were not. Competitive binding assays using these Mabs demonstrated the existence of two distinct antigenic regions (A and B) on the 40 Kd protein. Region A was recognized by group I Mabs and region B was by group II Mabs. The binding reaction with group I Mabs was affected by denaturing of the viral proteins, indicating that the antigenic region involving neutralization was conformation-dependent. The results of enzyme-linked immunosorbent assays and virus neutralization tests suggested that group I Mabs might react with one epitope within region A and group II Mabs with 2 or 3 epitopes within region B.     

Epitopes on the major capsid protein of simian virus 40

Thirteen monoclonal antibodies which react with the major capsid protein (VP1) of simian virus 40 (SV40) have been isolated. Of these, five neutralized viral infectivity when added in sufficient concentration. Seven of the antibodies reacted with denatured VP1 and also recognized fragments generated by protease or cyanogen bromide cleavage. The region of VP1 recognized by all seven antibodies was mapped within a nine-amino-acid segment located in the carboxyl portion of the protein (from amino acid positions 312 to 321). This region is likely to protrude from the surface of the protein as judged by high hydrophilicity and low hydropathy predicted from the amino acid sequence and lack of secondary structure by contrast with the rest of the protein for which predominantly beta-sheet structure is predicted. Competition between these antibodies and synthetic peptides for binding to virus particles confirmed that the continuous epitope is contained within the nine-amino-acid sequence. Competition between the different monoclonal antibodies suggested that the continuous epitope was also part of more complex discontinuous epitopes recognized by some of the other antibodies. These results support a model in which a segment of the carboxyl-terminal portion of VP1 protrudes from the surface of the virus to form an antigenic structure.     

Localization of conserved and nonconserved epitopes within the Rous sarcoma virus-encoded src protein.

We investigated the location of binding sites of pp60src-specific monoclonal antibodies by immunoprecipitating a panel of structurally altered src proteins. Two families of antibodies which recognized epitopes mapping to either amino acid residues 28 to 38 or 92 to 128 were identified. The highly conserved nature of the epitope defined by residues 92 to 128 suggests that it may represent an important functional region of the cellular src protein.     

Identification of two novel B-cell epitopes on the nucleocapsid protein of porcine deltacoronavirus

Highlights
1. Two monoclonal antibodies against newly emerged porcine deltacoronavirus nucleocapsid protein were prepared.
2. The epitopes that these two monoclonal antibodies recognized on nucleocapsid protein were identified.
3. The monoclonal antibody 6B7 recognized a linear epitope of N protein, while the 7F8 recognized a conformational epitope.
4. Conservation of the identified epitopes between different coronaviruses was analyzed.     

Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.     

Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties

Several novel anti-CD20 monoclonal antibodies are currently in development with the aim of improving the treatment of B cell malignancies. Mutagenesis and epitope mapping studies have revealed differences between the CD20 epitopes recognized by these antibodies. Recently, X-ray crystallography studies confirmed that the Type I CD20 antibody rituximab and the Type II CD20 antibody obinutuzumab (GA101) differ fundamentally in their interaction with CD20 despite recognizing a partially overlapping epitope on CD20. The Type I CD20 antibodies rituximab and ofatumumab are known to bind to different epitopes. The differences suggest that the biological properties of these antibodies are not solely determined by their core epitope sequences, but also depend on other factors, such as the elbow hinge angle, the orientation of the bound antibody and differential effects mediated by the Fc region of the antibody. Taken together, these factors may explain differences in the preclinical properties and clinical efficacy of anti-CD20 antibodies.