Fine Specificity Of Antibodies Research Articles

Understanding and Modulating Antibody Fine Specificity: Lessons from Combinatorial Biology

Combinatorial biology methods such as phage and yeast display, suitable for the generation and screening of huge numbers of protein fragments and mutated variants, have been useful when dissecting the molecular details of the interactions between antibodies and their target antigens (mainly those of protein nature). The relevance of these studies goes far beyond the mere description of binding interfaces, as the information obtained has implications for the understanding of the chemistry of antibody–antigen binding reactions and the biological effects of antibodies. Further modification of the interactions through combinatorial methods to manipulate the key properties of antibodies (affinity and fine specificity) can result in the emergence of novel research tools and optimized therapeutics.

Molecular Simulations Reveal the Role of Antibody Fine Specificity and Viral Maturation State on Antibody-Dependent Enhancement of Infection in Dengue Virus.

Recent clinical studies have revealed that severe symptoms of dengue fever are associated with low pre-existing antibody levels. These findings provide direct clinical evidence for the theory of antibody-dependent enhancement of infection (ADE), which postulates that sub-neutralizing levels of antibodies facilitate the invasion of host cells by the dengue virus. Here, we carried out molecular simulations guided by previous in vitro experiments and structural studies to explore the role of antibody fine-specificity, viral conformation, and maturation state—key aspects of dengue virology that are difficult to manipulate experimentally—on ADE in the context of primary and secondary infections. Our simulation results reproduced in vitro studies of ADE, providing a molecular basis for how sub-neutralizing antibody concentrations can enhance infection. We found that antibody fine specificity, or the relative antibody response to different epitopes on the surface of the dengue virus, plays a major role in determining the degree of ADE observed at low antibody concentrations. Specifically, we found that the higher the relative antibody response to certain cross-reactive epitopes, such as the fusion loop or prM, the greater was the range of antibody concentrations where ADE occurred, providing a basis for why low antibody concentrations are associated with severe dengue disease in secondary infections. Furthermore, we found that partially mature viral states, in particular, are associated with the greatest degree of ADE.

Dengue virus antibody database: Systematically linking serotype-specificity with epitope mapping in dengue virus.

BackgroundA majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response.Methods/Principal findingsWe hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III.Conclusions/SignificanceOur results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response in terms of cross-reactivity, and a focusing of the response to a subset of epitopes, including some, such as the fusion-loop region, that are implicated in poor neutralization and antibody-dependent enhancement of infection.

How specific is too specific? B‐cell responses to viral infections reveal the importance of breadth over depth

Influenza virus infection induces robust and highly protective B-cell responses. Knowledge gained from the analysis of such protective humoral responses can provide important clues for the design of successful vaccines and vaccination approaches and also provides a window into the regulation of fundamental aspects of B-cell responses that may not be at play when responses to non-replicating agents are studied. Here, I review features of the B-cell response to viruses, with emphasis on influenza virus infection, a highly localized infection of respiratory tract epithelial cells, and a response that is directed against a virus that continuously undergoes genetic changes to its surface spike protein, a major target of neutralizing antibodies. Two aspects of the B-cell response to influenza are discussed here, namely polyreactive natural antibodies and the role and function of germinal center responses. Both these features of the B-cell response raise the question of how important antibody fine-specificity is for long-term protection from infection. As outlined, the pathogenesis of influenza virus and the nature of the antiviral B-cell response seem to emphasize repertoire diversity over affinity maturation as driving forces behind the influenza-specific B-cell immunity.

Isolation and characterization of human polyreactive pneumococcal polysaccharide antibodies

Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.

Fine specificity of antibodies against AQP4: Epitope mapping reveals intracellular epitopes

The autoantibody to aquaporin-4 (AQP4) is a marker and a pathogenetic factor in Neuromyelitis Optica (NMO) (Devic’s syndrome). Our aim was to identify B-cell antigenic linear epitopes of the AQP4 protein and investigate similarities with other molecules. To this end, we screened sera from 21 patients positive for anti-AQP4 antibodies (study group), from 23 SLE and 23 pSS patients without neurologic involvement (disease controls) and from 28 healthy individuals (normal controls). Eleven peptides, spanning the entire intracellular and extracellular domains of the AQP4 molecule, were synthesized, and all sera were screened for anti-peptide antibodies by ELISA. Specificity was evaluated by homologous inhibition assays. NMO positive sera exhibited reactivity against 3 different peptides spanning the sequences aa1–22 (AQPpep1) (42.9% of patients), aa88–113 (AQPpep4) (33%) and aa252–275 (AQPpep8) (23.8%). All epitopes were localized in the intracellular domains of AQP4. Homologous inhibition rates were ranging from 71.1% to 84.3%. A 73% sequence homology was observed between AQPpep8′ aa257–271, a 15-mer peptide part of the AQPpep8 aa252–275, and the aa219–233 domain of the Tax1-HTLV-1 binding protein (TAX1BP1), a host protein associated with replication of the Human T-Lymphotropic Virus 1 (HTLV-1). Antibodies against the AQP4 and the TAX1BP1 15-mer peptides were detected in 26.3% (N = 5) and 31.6% (N = 6) of NMO positive sera (rs = 0.81, P < 0.0001). Healthy controls did not react with these peptides, while homologous and cross-inhibition assays confirmed binding specificity. This first epitope mapping for AQP4 reveals that a significant proportion of anti-AQP4 antibodies target linear epitopes localized in the intracellular domains of the channel. One of the epitopes displays high similarity with a portion of TAX1BP1 protein.

Anti-GM1 IgG antibodies in Guillain–Barré syndrome: fine specificity is associated with disease severity

BackgroundClinical severity of Guillain–Barré syndrome (GBS) is highly variable, but the immunopathological reason is unknown.ObjectiveThe study was designed to show which antibody parameters are associated with disease severity in GBS...

Phage Displayed scFv: pIII Scaffold May Fine Tune Binding Specificity

The fine specificity of antibodies is important for their discriminating powers during diagnostics and in vivo therapy. We have attempted to isolate human scFv antibodies to the oncofetal antigen, the placental isozyme of alkaline phosphatase (PLAP) in which it is important to distinguish between the closely related intestinal alkaline phosphatase (IAP) and bone alkaline phosphatase (BAP) isozymes. As the antibodies are selected in the phage displayed form and might be finally used as different entities, including the soluble scFv form, it may be important to look at the influence of scaffolds in determining specificity. There have been earlier reports of the role of the constant region and other scaffolding proteins in determining specificity. In this paper, we report isolation of one such clone, E6, which showed specificity to PLAP in phage antibody form but lost the specificity when soluble scFv was tested for same, and showed partial cross reactivity to BAP. We suggest that the altered specificity of scFv might be the result of loss of phage pIII scaffold, which is present in phage-displayed antibody and may help the displayed antibody to assume specific conformational structure, which may govern binding characteristics of the same.

Fine specificity of antibodies against phospholipids and beta-2-glycoprotein I in monoclonal gammopathy associated neuropathies

Phospholipids are abundantly represented within the nervous system. Aim of our study was to evaluate the presence and fine specificity of anti phospholipid antibodies (aPLAb) among patients with monoclonal gammopathy associated neuropathy. Thirty nine percent of these patients had high titers of aPLAb, mostly associated with low levels of anti beta2 glycoprotein I, which suggests different antibody specificity compared to patients with anti phospholipid syndrome. Further 6/48 patients with dysimmune neuropathy without monoclonal gammopathy had positive aPLAb titers. APLAb strongly cross-reacted with sulfatide. These findings suggest an adjuntive role of aPLAb on nerve damage and may help to better understand the nerve binding properties of anti-sulfatide antibodies.

Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure.

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x indicates the identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions in the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 in the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 in an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was indicated by marked differences in their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts.

Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens

Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

Fine epitope specificity of antibodies to region II of the Plasmodiumvivax circumsporozoite protein correlates with ability to bind recombinant protein and sporozoites

Recent work has suggested that important B- and T-cell epitopes on the circumsporozoite protein (CSP) of Plasmodium vivax lie external to the major repeat regions of the protein. We have studied two naturally exposed human populations (Caucasian and Papua New Guineans) and determined the antibody response to yeast-derived recombinant CSPs, overlapping synthetic peptides spanning amino acids 76–348 of the Belem P. vivax CSP and overlapping peptides representing the variant repeats of the VK247 strain of P. vivax. We have demonstrated that the P. vivax CSP-specific antibody response is directed towards areas within the repeat region as well as areas external to this; but the dominant epitopes recognized by the two populations studied, were distinct. One epitope, lying external to the repeats and recognized by both populations, partially overlaps an area of the protein referred to as region II-plus. Sera from malaria-exposed Papua New Guineans and Thais contained antibodies to this epitope (V22, single letter amino acid sequence TCGVGVRVRRRVNAANKKPE) which were capable of recognizing sporozoites, as determined by quantitative inhibition IFA. Seventeen percent of PNG sera had antibodies to this peptide compared with 33% who had antibodies to the central repeats of the protein. Immunization of mice with recombinant CSP did not induce antibodies to V22. However, immunization with overlapping peptide epitopes representing this region (V21 or V22) induced specific antibodies but only two sera recognized both V21 and V22 and, by inference, the overlapping peptide sequence (TCGVGVRVRR). Antibodies in these two sera could bind recombinant CSP in ELISA; however, in contrast, nine sera which recognized either V21 or V22 alone did not bind CSP. Only one of two sera containing antibodies recognizing CSP stained P. vivax sporozoites. This serum also recognized an epitope dependent upon two amino acids aminoterminal to V22. These data suggest that the fine specificity of antibodies is a critical determinant for binding to both recCSP and sporozoites.

Characterisation of the Antibody Response to the Extracellular Region of Recombinant Thyrotropin Receptor

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable” of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.

Of four murine, anti- Shigella itdysenteriae type 1 O-polysaccharide antibodies, three employ V-genes that differ extensively from those of the fourth

Three murine, monoclonal antibodies, IgM 5286 F2, IgM 5297 Cl, and IgG 5338 H4 were generated against Shigella dysenteriae type 1 O-specific polysaccharide (O-SP)-conjugate. They are specific for the O-SP, which is a poly-[α- l-rhamnopyranosyl-(1→3)-α- l-rhamnopyranosyl-(1→2)-α- d-galactopyranosyl-(1→3)-2-deoxy-2-amino- N-acetyl-α- d- glucopyranosyl]. The VH and VL genes of these antibodies were cloned and their sequences determined. They showed 93% homology, but were quite different to the primary sequence of IgM 3707 E9, of the same O-SP-specificity, previously reported. The fine-specificities of both IgG 5338 H4 and IgM 3707 E9 were for the same disaccharide moiety in the O-SP, while IgMs 5286 F2 and 5297 C1 showed fine-specificity for the entire repeating unit of the O-SP. Therefore, divergent sequences can confer upon antibodies similar-, or even identical-carbohydrate-epitope fine-specificity. In addition, close primary sequence-homology does not preclude differences in antibody fine-specificity.

B-cell repertoire selection: Fine specificity of antibodies elicited by bacterial polysaccharide dextrans and by V-region reactive ligands

B-cell repertoire selection: Fine specificity of antibodies elicited by bacterial polysaccharide dextrans and by V-region reactive ligands

Comprehensive Epitope Analysis of Monoclonal Anti-proenkephaliln Antibodies using Phage Display Libraries and Synthetic Peptides: Revelation of Antibody Fine Specificities Caused by Somatic Mutations in the Variable Region Genes

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with the help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody sites.It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VHchain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity.

Fine specificities of antibodies in sera and cerebrospinal fluid in herpes virus infections of the central nervous system as detected by the antigen variable immunoblot technique

Applying the immunoblot technique a sensitive and specific method was developed for the detection of intrathecally synthesized antibodies against individual specific proteins that are antigens of various infectious agents causing encephalitis. Paired serum and cerebrospinal fluid (CSF) samples from five patients with herpes virus infections of the central nervous system (CNS) (three herpes simplex virus encephalitis, one varicella zoster virus encephalitis, one zoster ganglionitis) were investigated for the presence of locally produced IgG against the electrophoretically separated antigens of herpes simplex virus (HSV), varicella zoster virus (VZV) and human cytomegalovirus (HCMV), as well as for IgM antibodies in one case of HSV encephalitis. In two cases (HSV encephalitis and VZV encephalitis) four and one antibody, respectively, were found that were synthesized intrathecally only. In the other cases the patterns of sera and CSF antibodies were similar, the CSF antibodies showing an all-over stronger reaction, at identical IgG concentrations. IN contrast to the conception of a ‘limited heterogeneity’ of intrathecal antibody synthesis in encephalitis, we thus found an ‘ expanded heterogeneity’ of the intrathecally sythesized antibodies in comparison to the corresponding serum antibodies.

Molecular analysis and fine specificity of antibodies against an organophosphorus hapten.

We have identified four fine specificity groups reactive with the organophosphorus hapten Soman among 46 hybridomas generated in specific response to immunization with Soman-keyhole limpet hemocyanin (KLH). The different fine specificity groups do not appear to correlate with the use of particular V genes. Molecular analysis of VH genes demonstrates predominant use of VH J558 family members in hybridomas of all fine specificity groups although several different VH genes within this family as well as others are able to contribute. Diversity of VH gene usage was also apparent in primary IgM-producing hybridomas. In contrast, there appears to be restricted L chain usage; a large number (18/46) used the V kappa 1 family. Interestingly, the V kappa 1 family also plays an important role in the memory response to phosphocholine (PC)-KLH, a related organophosphate hapten which shares several structural features with Soman, particularly when coupled to protein carriers. The V kappa 1 C gene appears to predominate in the PC-KLH response. Restriction analysis of DNA from the V kappa 1-positive Soman-KLH-specific hybridomas suggests that a single V kappa 1 gene may be utilized by 17/18 but that this gene is different from V kappa 1 C and may be V kappa 1 A. We propose that members of the V kappa 1 family contribute favorably in generating combining sites that recognize all or part of the structural features shared by the two haptenic structures Soman and PC when they are coupled to protein. This most likely involves recognition of the phenyl linker moiety as the dominant feature. It appears that the L chain rather than the H chain may play a more significant role in forming the phenyl-Soman-specific combining site and perhaps the combining sites for phenyl or ring structures in general.

Influence of Antibody Fine Specificity on Development of Myasthenia in Mice after Passive Transfer of Anti‐AChR

Influence of Antibody Fine Specificity on Development of Myasthenia in Mice after Passive Transfer of Anti‐AChR

Two distinct but overlapping antibody binding sites in the pre-S(2) region of HBsAg localized within 11 continuous residues.

The fine specificity of the humoral immune response to the pre-S(2) region of the hepatitis B surface antigen was studied. It was demonstrated that the murine antibody response to the pre-S(2) region is focused on residues 133 through 143, and two distinct but overlapping epitopes were identified within 11 continuous residues. One epitope, defined by p133-139, is group specific, and the other epitope, defined by p137-143, is influenced by a subtype-dependent amino acid substitution at residue 141. However, the influence of residue 141 was "covert" in that it was only detected when synthetic antigens of 19 amino acids or smaller were used as the solid-phase ligand. The minimum size of both epitopes (p133-139 and p137-143) was seven amino acids. The physical and chemical form of the immunogen (i.e., protein vs peptide; conjugated vs free peptide) influenced antibody fine specificity. In quantitative antibody inhibition studies it was demonstrated that antibodies with nonoverlapping as well as overlapping fine specificities were capable of mutual inhibition. Finally, human HBV-infected, patient sera were shown to possess anti-pre-S(2) region antibodies that recognized sequences in common with the murine antisera. These results have implications relevant to the design of synthetic and recombinant second generation HBV vaccines and diagnostic reagents.