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PubMed1996

Design and synthesis of a highly immunogenic, discontinuous epitope of HIV-1 gp120 which binds to CD4+ve transfected cells.

Here we report the design and synthesis of a novel 32-mer peptide, Lys364-378Val445-459.oxidized (named GC-1), which represents a discontinuous epitope from the C3 and C4 domains of gp120 from the HIV-1 IIIB isolate. This peptide induces high titre IgG antibody responses in mice, indicating that it has both B and T cell epitopes. Epitope mapping using reduced GC-1 and appropriate linear peptides demonstrated that a large proportion of the antibodies raised in mice were directed against discontinuous epitope(s). Furthermore, antibodies to GC-1 peptide cross-reacted with purified HIV-1 strain IIIB gp120, indicating the GC-1 mimicked at least one epitope of the native protein. The peptide, which incorporates three gp120 residues Asp 368, Glu 370 and Asp 457, previously shown to be critical for CD4 ligation, bound to the surface of a CD4 transfected human epithelial cell line HeLa, but not to the parent cell line and inhibited binding of recombinant HIV-1 gp120 to recombinant soluble CD4. We have synthesized the first of a series of discontinuous peptides which will be useful for the probing of interactions of HIV-1 gp120 with the CD4 molecule.

Amino Acid Sequence↗
PubMed2000

Human immune response to streptococcal inhibitor of complement, a serotype M1 group A Streptococcus extracellular protein involved in epidemics.

Streptococcal inhibitor of complement (Sic) is a highly polymorphic extracellular protein made by serotype M1 group A Streptococcus strains that contributes to bacterial persistence in the mammalian upper respiratory tract. New variants of the Sic protein arise very rapidly by positive selection in human populations during M1 epidemics. The human antibody response to Sic was analyzed. Of 636 persons living in diverse localities, 43% had anti-Sic serum antibodies, but only 16.4% had anti-M1 protein serum antibody. Anti-Sic antibody was also present in nasal wash specimens in high frequency. Linear B cell epitope mapping showed that serum antibodies recognized epitopes located in structurally variable regions of Sic and the amino terminal hypervariable region of the M1 protein. Phage display analyses confirmed that the polymorphic regions of Sic are primary targets of host antibodies. These results support the hypothesis that selection of Sic variants occurs on mucosal surfaces by a mechanism that involves acquired host antibody.

Adolescent↗
PubMed1995

Immunogenicity of recombinant Plasmodium falciparum rhoptry associated proteins 1 and 2.

Mice and rabbits immunized with recombinant forms of malaria vaccine candidate antigens rhoptry-associated proteins 1 and 2 (RAP-1, RAP-2 and rRAP-1, rRAP-2) produce antibodies at titres equivalent to monoclonal antibody ascites fluid raised against the native proteins. Sera from animals immunized with rRAP-1 contain antibodies which recognize the native protein by indirect immunofluorescence and immunoblotting, partially inhibit erythrocyte invasion in vitro and are long lasting. Epitope mapping shows these antibodies predominantly recognize epitopes in the N-terminal third of rRAP-1, some of which coincide with the targets of inhibitory monoclonal antibodies. By contrast, sera from animals immunized with rRAP-2 contain antibodies which recognize the recombinant but not the native protein.

Amino Acid Sequence↗
PubMed1985

Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing.

A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary-shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co-linearly with the peptide chain.

Amino Acid Sequence↗
PubMed2002

Epitope analysis of antibodies in Japanese to human cytomegalovirus phosphoprotein 150 with synthetic peptides.

Serological detection of antibodies specific to human cytomegalovirus (HCMV) is not reliable because the assay uses the whole HCMV protein fraction. Antigenic materials composed of well-characterized viral proteins are being tried for serodiagnosis in Europe. Epitopes of antibodies to HCMV phosphoprotein 150 (pp150) encoded by UL32 in Japanese individuals were investigated for comparison with the results in Europe. The major epitopes on amino acid residues 496 to 652 of HCMV pp150 were identified and the detection of antibodies with an enzyme-linked immunosorbent assay (ELISA) of synthetic peptides against the main epitopes was established. Fifteen seropositive and five seronegative serum samples for the epitope mapping and 131 seropositive and 50 seronegative samples for ELISA were investigated. Overlapping 15-mer peptides moving by two amino acids through V496-H652 were synthesized. The main epitope regions were V508-D530, L526-Q544, S536-D554, T616-G634, S624-P642, and L632-H652. When each peptide was conjugated with bovine serum albumin for ELISA, 80.9% of the seropositive samples were judged to be positive. The results of this study are the same as those for European sera, so the antigenic materials developed in Europe might be used to replace the whole HCMV protein fraction in Japanese.

Amino Acid Sequence↗
PubMed2003

Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level.

Due to IgE cross-reactivity, birch pollen-allergic individuals frequently develop type I hypersensitivity reactions to celery tuber. We evaluated the T cell response to the major allergen in celeriac, Api g 1, and the cellular cross-reactivity with its homologous major allergen in birch pollen, Bet v 1. Api g 1-specific T cell lines (TCL) and clones (TCC) were established from peripheral blood mononuclear cells of allergic patients. Epitope mapping of Api g 1 with overlapping Api g 1-derived peptides revealed one dominant T cell-activating region, Api g 1(109-126). TCL and TCC generated with Api g 1 cross-reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet v 1 were stronger than to Api g 1. Epitope mapping with Bet v 1-derived peptides revealed that T cells specific for several distinct epitopes distributed over the complete Bet v 1 molecule could be activated by Api g 1. Bet v 1(109-126) was identified as the most important T cell epitope for cross-reactivity with Api g 1. This epitope shares 72% amino acid sequence similarity with the major T cell-activating region of the food allergen, Api g 1(109-126). Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross-reactivity with the major birch pollen allergen. The activation of Bet v 1-specific Th2 cells by Api g 1, in particular outside the pollen season, may have consequences for birch pollen-allergic individuals.

Allergens↗
PubMed2005

Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides.

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.

Actinomycetales Infections↗
PubMed2000

CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor.

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.

Alternative Splicing↗
PubMed1997

Cell cycle changes in A-type lamin associations detected in human dermal fibroblasts using monoclonal antibodies.

A new panel of anti-A-type lamin monoclonal antibodies was generated. Epitope mapping was performed by immunoblotting against GST-lamin fusion peptides. Epitopes were mapped to four different regions of human lamin A and three different regions of human lamin C. The distribution of A-type lamins was compared with the distribution of the proliferation marker Ki67 in proliferating and quiescent cultures of human dermal fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stained the nuclear envelope of proliferating and quiescent cells equally brightly. In contrast, antibodies recognizing epitopes in the head domain and rod domain of lamins A and C and the tail domain of lamin A stained the nuclear envelope of quiescent cells strongly but reacted poorly or not at all with the nuclear envelope of proliferating cells. Changes in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and this occurred by two distinct mechanisms. Epitope masking in the head domain of lamins A and C occurred as a result of protein phosphorylation. Epitope masking in the rod domain of lamins A and C and in the tail domain of lamin A occurred through a physical association between the lamin and chromatin and/or other nuclear proteins. The cell cycle timing of epitope masking was investigated in HDFs that had been restimulated after serum starvation. Extensive epitope masking in restimulated cells only occurred after cells had passed through mitosis. These results are consistent with the hypothesis that rearrangement of A-type lamin filaments, as cells progress from a quiescent to a proliferating state, results in altered lamina associations.

Animals↗
PubMed2005

The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides.

We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal alpha4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity.

Amino Acid Sequence↗
PubMed1997

Searching for pathogenic epitopes in thyroglobulin: parameters and caveats.

Last year marked the 40th anniversary of the discovery that thyroglobulin (Tg) is a major autoantigen in autoimmune thyroiditis. The Tg molecule presents unique challenges for epitope mapping owing to its large size and extensive iodination. Consequently, pathogenic determinants have only recently been identified. Here, George Carayanniotis and Varada Rao summarize the approaches used to determine pathogenic Tg T-cell epitopes and discuss caveats in this unusual quest.

Animals↗
PubMed2000

Identification of a major B-cell epitope of the Plasmodium falciparum glutamate-rich protein (GLURP), targeted by human antibodies mediating parasite killing.

The antigenicity of the glutamate-rich protein (GLURP) of Plasmodium falciparum was comprehensively evaluated in epitope-mapping studies utilizing a phage display library, synthetic peptides and anti-GLURP IgG preparations previously shown to promote strong antibody-dependent cellular inhibition (ADCI) effects. We identified six major B-cell epitopes within the nonrepetitive region R0, corresponding to amino acid residues 173 to 187 (P1), 193 to 207 (P3), 216 to 229 (P4), 264 to 288 (P11), 343 to 357 (P10), and 407 to 434 (S3). Of these, four (P1, P3, P4, and S3) were frequently recognized by high-titered IgG antibodies in plasma samples from immune Liberian adults (prevalence: 29.1-45.0%). The three epitopes P1, P3, and P4 contained a common motif (seven out of nine positions are identical) and may thus constitute a family of structurally related epitopes. This leaves two distinct epitopes, one (P3) representing this new epitope family and S3 as targets for biologically active antibodies. Human IgG antibodies from single plasma samples were affinity-purified against these peptides. P3-specific IgG preparations were consistently more effective in ADCI than S3-specific IgG. Among the different GLURP epitopes, we therefore suggest that the P3 epitope is potentially the most important epitope in GLURP for the development of clinical immunity to malaria in man.

Adult↗
PubMed1990

Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies.

Hepatitis delta virus (HDV) is a defective RNA virus which is dependent on hepatitis B virus for essential helper functions. Only a single highly basic phosphoprotein, HDV antigen (HDAg), is expressed by the HDV genome during infection in humans. Antibody directed to HDAg is important in the diagnosis of HDV infection, and it is likely but not yet proven that the immune response to HDAg provides significant protection against subsequent exposures to HDV. In an effort to map the antigenic domains of HDAg, 209 overlapping hexapeptides, spanning the entire 214 amino acid residues of the protein, were synthesized on polyethylene pins and probed by enzyme-linked immunosorbent assay with sera containing high titers of anti-HD antibodies. Domains recognized by antibodies present in serum from human chronic carriers of this virus included residues 2 to 7, 63 to 74, 86 to 91, 94 to 100, 159 to 172, 174 to 195, and 197 to 207. Antibody from an acutely superinfected woodchuck recognized similar epitopes, as well as a domain spanning residues 121 to 128. Together, residues in these antigenic domains constitute 41% of the HDAg molecule. Oligopeptides 15 to 29 residues in length and representing epitopes of HDAg found to be dominant in humans (residues 2 to 17, 156 to 184, and 197 to 211) were synthesized in bulk and found to possess significant antigenic activity by microdilution enzyme-linked immunosorbent assay. The reactivity of peptide 197-211 with human sera confirms that the entire 214 amino acids of HDAg are expressed during infection in vivo. In addition, these results suggest that synthetic peptides may be useful reagents for development of new and improved diagnostic tests for HDV infection.

Amino Acid Sequence↗
PubMed2004

Progress made towards the development of a CMV peptide vaccine.

Cytomegalovirus disease still remains a major cause of morbidity and mortality in hematopoietic stem cell transplantation recipients. The cell-mediated immune response is essential in the maintenance of latency and the resolution of primary infections. The identification of immunodominant cytomegalovirus antigens has enabled researchers to determine the best candidate antigens to be included in a cytomegalovirus vaccine. Such a vaccine would have to stimulate both a cell-mediated and humoral immune response. Recent advances have enabled the rapid identification of minimal cytotoxic epitopes required to trigger such responses. Epitope mapping to date has mainly focused on the pp65 antigen but other antigens such as IE1 are starting to be mapped. A human leukocyte antigen allele hierarchy is starting to emerge that is dependent on the alleles present in an individual; this is relevant when considering what peptides should be included in a vaccine. This review looks at the current methods available for epitope mapping and the progress that has been made to date.

Algorithms↗
PubMed2004

Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity.

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFbeta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0-160 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.

Amino Acid Sequence↗
PubMed1995

Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies.

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.

Animals↗
PubMed1995

A 10-amino-acid linear sequence of VP1 of foot and mouth disease virus containing B- and T-cell epitopes induces protection in mice.

The area of foot and mouth disease virus (FMDV) comprising residues 140 and 160 of capsid protein VP1 has been used extensively as an immunogen in natural and experimental hosts. A detailed epitope mapping of this region, however, has not been reported. For this purpose a synthetic peptide containing the residues 135 to 160 (p135-160) of VP1 of FMDV O1 Campos was analyzed for its T- and B-cell epitopes. The p135-160 is highly immunogenic, either by itself or coupled to a carrier protein (BSA), elicits a long-lasting neutralizing antibody response in mice, and provides solid protection against virulent challenge. By using a set of synthetic 10mer overlapping peptides, which cover the entire sequence 135-160 of VP1, we have shown that at least four discrete B epitopes are regularly distributed along the peptide. Although immunization with each of the 10mers coupled with BSA as a carrier protein induced peptide-specific antibody responses, individually none of the 10mers was able to induce neutralizing antibodies. However, anti-135-160 antibodies sorted by immunoaffinity chromatography using each of the 10mers revealed the existence of at least four discrete neutralizing sites: one spanning residues 135-144, at least two more between residues 140 and 154, and another in the region 150-160. Moreover, T-cell epitopes were identified, both by antigen-dependent proliferation assays and by adoptive cell transfer. By both methods, a T-cell epitope was located in the area comprising residues 135-144; the cell transfer experiment, which seems to be more sensitive, also identified a second T-cell epitope between residues 150 and 160. Interestingly, when the region 135-144, which contains both B- and T-cell epitopes, was in a tandem repeat configuration it induced a strong neutralizing antibody response in mice and solid protection against the challenge.

Amino Acid Sequence↗
PubMed2005

The epitope recognized by a monoclonal antibody in influenza A virus M2 protein is immunogenic and confers immune protection.

Based on our previous study that the monoclonal antibody (mAb) 8C6 recognizing the extracellular domain of influenza A virus M2 protein (M2e) could passively induce protective immunity in mice, epitope mapping was performed in this study. A series of eight mutated M2e were constructed and expressed. It was found in immunoblotting assay that 8C6 could not bind to two of the mutated M2e proteins, which suggested the VETPIR epitope (aa7-12) in M2e for mAb 8C6. More important, EVETPIRN-peptide (aa6-13) conjugated immunogen induced high M2e specific antibody titer (1:25,600) in mice after booster immunization, and provided mice significantly 40% higher survival rate compared with the control group in challenge assay (p=0.0215), which suggested the EVETPIRN-epitope was immunogenic and actively conferred immune protection to some extent. In addition, sequence comparison suggested the EVETPIRN sequence (aa6-13) was highly conserved in all human influenza A strains. All these data suggested the EVETPIRN sequence in M2e could be one new target for influenza vaccine design.

Amino Acid Sequence↗