A flexible peptide linker enhances the immunoreactivity of two copies HBsAg preS1 (21-47) fusion protein

PreS1 (21-47) region of HBV large surface protein is hepatocyte receptor binding site and the anti-preS1 (21-47) antibody possesses the virus-neutralizing activity and protective effect. It is important to obtain the peptide with higher immunoreactivity on a large scale for detecting the anti-preS1 (21-47) antibody in the sera from HBV infected patients and future vaccine recipients. The expression vector pGEX SLS, which expressed two copies of the preS1 (21-47) peptide connected by a flexible linker (Gly4Ser3) fused to glutathione S-transferase (GST), was constructed. The fusion protein, named GST-SLS, was highly expressed in E. coli and purified by affinity chromatography. Ninety milligrams purified protein can be obtained from 1l of culture. The data in ELISA analysis showed that the immunoreactivity of GST-SLS was enhanced significantly in comparison with GST-S II, a GST fusion protein with two copies preS1 (21-47) linked directly; GST-S I, another GST fusion protein with one copy preS1 (21-47) and preS1 (21-47) synthesized peptide. In addition, GST-SLS has been tried to use in detecting anti-preS1 (21-47) antibody in the sera from HBV infected patients and a satisfied result was gained. Therefore, GST-SLS may have potential to be developed into a new kit for diagnosis and prognosis of hepatitis B (HB) patients.     

Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.     

修饰的含前S1、前S2免疫决定簇乙肝表面抗原融合多肽在毕赤酵母中的共表达

用BamHⅠ和BglⅡ双酶切含SS2嵌合基因片段的重组质粒pAO815-SS2,分离含AOX1启动子区、SS2嵌合基因和AOX1转录终止序列的表达盒。将分离的BamHⅠ-BglⅡ表达盒与经BamHⅠ线性化的pAO815-SS1质粒连接,转化大肠杆菌Top10F′,提取质粒,用BamHⅠ和BglⅡ双酶切分析重组质粒并筛选正向插入SS2表达盒的重组子。将该重组质粒用电转化法导入PichiapastorisGS115细胞,经表型筛选、小试表达和产物鉴定,构建了重组表达菌株GS115-SS1S2。重组菌株经甲醇诱导后制备抗原粗提液,进一步进行ELISA和Westernblot鉴定。ELISA结果显示,产物同时具有S、前S1和前S2抗原性。Westernblot分析进一步表明,表达产物既能与S抗体结合,也能与前S1和前S2抗体结合。工程菌经高密度发酵,表达量达到300~600mg/L。抗原经纯化后进行电镜观察,形成直径20~35nm的球形颗粒。纯化抗原经SDS-PAGE分析,SS1和SS2多肽仍然保持完整,基本无降解。     

Determination of the protective effects of neutralizing anti-hepatitis B virus (HBV) immunoglobulins by epitope mapping with recombinant HBV surface-antigen proteins

Anti-hepatitis B virus (HBV) surface-antigen immunoglobulins prepared from human sera are clinical reagents which have been approved for prophylactic treatment in HBV-exposed persons. The passive immunoprophylaxis with immunoglobulins is meant to cross-link viral particles, which are then further cleared by the host's own immune system. While antibodies specific for both anti-S- and anti-preS proteins have been proved to serve as effective anti-viral agents, so far the fine antigen specificity of clinical immunoglobulin preparations has not been determined. Using recombinant proteins covering the hepatitis B surface antigen, in the present study, the specificity of a commercially available immunoglobulin preparation was determined and immunodominant epitopes were mapped. Here, it is shown that the major reactivity of anti-HBV immunoglobulins is directed against the S-protein, and that no reactivity to the preS2 but a weak binding activity to the preS1 region was detectable. The antigen reactivity within the preS1 region was biased to the C-terminal region, which indicates the presence of a putative B-cell epitope. The evaluation of the antigen specificity and determination of novel protective epitopes will provide valuable information for the further development and improvement of prophylactic HBV immunoglobulins.     

Molecular analysis of the modulatory factors of the response to HBsAg in mice as an approach to HBV vaccine enhancement

Abstract Mice were injected with immune complexes containing the recombinant hepatitis B surface antigen (HBsAg) vaccine (S + preS2) bound to different monoclonal antibodies (mAbs), in order to determine whether an enhancement of the response to a human vaccine could be obtained and observed. Enhancement and indifference were observed, as well as a decrease in immunogenicity. N relationship could be established between any effect and affinity or isotypy of the bound mAbs. The preS2 region was rendered more immunogenic when an IgG2a mAb was bound to the S region of the HBsAg. The response to the S region was not modulated, whereas immunogenicity of the preS2 collinear region was decreased by antibody shielding. The mAb which was the most efficient as an enhancer of the antibody response also increased binding of the complexed immunogen to antigen presenting cells. The binding of a human mAb to the sole S region, but not to the preS2 region, should be tested as a potentiating agent of the anti-preS2 human immune response.     

不同佐剂对preS2／S乙型肝炎疫苗免疫原性的影响

目的探讨不同佐剂对preS2／S乙型肝炎疫苗免疫原性的影响。方法将BALB／C小鼠随机分为6组：铝屯剂组（S＋AI和S2／S＋A1）、细胞因子佐剂组（S2／S＋CKl、S2／S＋CK2和S2／S＋CK3）及阴性对照组（NG）,用相应抗原且腔免疫各组小鼠,1mL／只,分别于免疫后2、4和6周采血,分离血清,ELISA法检测血清中抗-HBs抗体和preS2圭体水平;wsT一1法检测淋巴细胞增殖情况。结果免疫后2周,铝佐剂组（S＋A1和S2／S＋A1）抗体水平较高,阳转i达100％,而细胞因子佐剂组抗-HBs抗体水平均较低,S2／S＋CK2及S2／S＋CK3组抗体阳转率为0,与NG组比较2异无统计学意义（P〉0．05）;免疫后4周,细胞因子佐剂组抗-HBs抗体水平升高明显,铝佐剂组S2／S＋A1抗体水习显著高于S＋A1（P〈0．05）;免疫后6周,细胞因子佐剂组抗体水平高于铝佐剂组,其中S2／S＋CKl组抗体水平较高免疫后2周,各组小鼠血清中抗-preS2抗体均达到较高水平,各组间差异均无统计学意义（P〉0．05）;免疫后4月和6周,抗体水平均有所下降,但下降不明显;铝佐剂组S＋AI和S2／S＋A1抗-preS2抗体水平差异无统计学意义（P0．05）。免疫后4周,各组小鼠淋巴细胞刺激指数铝佐剂组（S＋A1和S2／S＋A1）低于细胞因子佐剂组,且差异有统t学意义（P〈0．05）,其中S2／S＋CKl组淋巴细胞增殖水平较高。结论不同佐剂的preS2／S乙型肝炎疫苗均能诱导较高水平的抗-HBs和抗-preS2抗体,细胞因子佐剂组淋巴细胞增殖水平显著高于铝佐剂组,为新型乙型肝炎疫l和治疗性乙型肝炎疫苗的研究提供了实验依据。     

乙型肝炎病毒前S1抗原(1-42)与核心抗原(1-144)在大肠杆菌中表达的融合蛋白CS1的免疫原性研究

对重组乙型肝炎（乙肝）病毒前S1抗原（1－42）及核心抗原（1－144）表达的融合蛋白CS1进行了免疫原性的研究分析,以便为探索HBV治疗性疫苗地研制提供实验依据。用脂质体转染的方法转染小鼠肥大细胞瘤细胞系P815,用乳酸脱氢酶的方法检测了该融合蛋白诱导小鼠的细胞毒T淋巴细胞（CTL）反应,用ELISA方法测定了小鼠的体液免疫反应。结果表明,用脂质体转染的方法建立了表达乙肝核心和前S1抗原的细胞系（P99－815）,该融合蛋白诱导出小鼠的细胞毒T淋巴细胞（CTL）反应,小鼠的体液免疫反应也较好。这为今后进一步探索慢性乙肝治疗性疫苗奠定了必要的基础。     

Genotyping of hepatitis B virus in Malaysia based on the nucleotide sequence of preS and S genes

Hepatitis B virus (HBV) has been classified into eight genotypes, designated A-H. These genotypes are known to have distinct geographic distributions. The clinical importance of genotype-related differences in the pathogenicity of HBV has been revealed recently. In Malaysia, the current distribution of HBV remains unclear. The aim of this study was to determine the genotypes and subtypes of HBV by using PCR, followed by DNA sequencing, as well as to analyse the mutations in the immunodominant region of preS and S proteins. The S gene sequence was determined from HBV DNA of four apparently healthy blood donors' sera and three sera from asymptomatic chronic hepatitis B carriers. Of this batch of sera, the preS gene sequence was obtained from HBV DNA from three out of the four blood donors and two out of the three chronic carriers. Due to insufficient sera, we had to resort to using sera from another blood donor to make up for the sixth DNA sequence of the preS gene. Based on the comparative analysis of the preS sequences with the reported sequences in the GenBank database, HBV DNA from two normal carriers was classified as genotype C. Genotype B was assigned to HBV from one blood donor and two hepatitis B chronic carriers, whereas HBV of one chronic carrier was of genotype D. Based on the S gene sequences, HBV from three blood donors was of genotype C, that of one blood donor and one chronic carrier was of genotype B, and the remaining, of genotype D. In the five cases where both preS and S gene sequences were determined, the genotypes assigned based on either the preS or S gene sequences were in concordance. The nature of the deduced amino acid (aa) sequences at positions 125, 127, 134, 143, 159, 161 and 168 of the S gene enabled the classification of these sequences into subtypes, namely, adrq+, adw2 and ayw2. The clustering of our DNA sequences into genotype groups corresponded to their respective subtype, that is, adw2 in genotype B, adrq in genotype C and ayw in genotype D. Analysis of the point mutations revealed that five of the sequences contained aa substitutions at immunodominant epitopes involved in B or/and T cell recognition. In conclusion, despite the low numbers of samples studied, due to budget constraints, these data are still worthwhile reporting, as it is important for the control of HBV infections. In addition, the genotype and mutational data obtained in this study may be useful for designing new treatment regimes for HBV patients.     

Behavior of a short preS1 epitope on the surface of hepatitis B core particles

The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79-88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79-93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79-81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune anti-HBc antibodies.     

Overexpression of hepatitis B virus surface antigens including the preS1 region in a serum-free Chinese hamster ovary cell line

Current hepatitis B virus (HBV) vaccines consist of preparations of recombinant HBV major surface antigen (sAg) and are protective in about 90-95% of vaccinated subjects. In improved vaccines, the frequency of nonresponders to the classical vaccine could be reduced by including additional epitopes from the preS-domains of the middle and large surface antigens. In this report, the development and characterization of a CHO cell line for HBsAg, expressing major, middle, and large antigens are described. Despite the previously reported retention of secreted proteins by the preS1 domain, cell lines could be amplified that secreted large amounts of the complete set of antigens. A producer line was established that expressed 1mg HBsAg per 100ml suspension culture per week during exponential growth. The productivity per cell increased further by at least threefold when the culture reached the stationary phase at high cell densities. In the production cell line, several hundred copies of the HBV vector were integrated at two adjacent sites into chromosome 2. The cell line was adapted to growth in a defined protein-free medium minimizing the risk of adventitious agents introduced by animal derived supplements. The cell line stably produced antigen over several months. In the candidate vaccine, both preS2 and preS1 domains were present at ratios similar to HBsAg from human sera. In summary, a production cell line for an improved HBV vaccine is presented with properties such as high productivity, long term stability of expression, and growth in protein-free media.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

Anti-pre-S2 antibody response in subjects vaccinated against hepatitis B and in naturally immunized subjects

Serum samples from individuals immunized with a pepsinized or non-pepsinized vaccine and from patients who had recovered from acute hepatitis B or who developed a chronic form of the disease, were analysed for the presence of antibody against the pre-S2 epitope of the hepatitis B virus.Anti-pre-S2 antibody was absent in all but one individual immunized with the pepsinized vaccine. Thirty-eight percent of the subjects who responded by anti-HBs production to the non-pepsinized preparation showed anti-pre-S2 antibody one year after complete vaccination. Among subjects who did not produce anti-HBs after immunization with this vaccine, 1 single individual produced anti-pre-S2 antibody. Anti-preS2 antibody was detectable after one year in 38% of the patients who recovered from acute hepatitis B, but in none of those with chronic hepatitis B. The kinetics of anti-pre-S2 antibody response to a booster injection was also analysed 1 month and 1 year after the 3rd injection and 1 month after the 4th injection of the non-pepsinized vaccine.     

Identification of the critical regions in hepatitis B virus preS required for its stability.

As a hepatitis B virus (HBV) envelope domain, preS plays significant roles in receptor recognition and viral infection. However, the regions critical for maintaining a stable and functional conformation of preS are still unclear and require further investigation. In order to unravel these regions, serially truncated fragments of preS were constructed and expressed in Escherichia coli. Their solubility, stability, secondary structure, and affinity to polyclonal antibodies and hepatocytes were examined. The results showed that amino acids 31-36 were vital for its stable conformation, and the absence of 10-36 amino acids significantly reduced its binding to polyclonal antibodies as well as hepatocytes. The most stable fragment 1-120 (preS1 + N-terminal 12 amino acids of preS2), perhaps the core of preS, was discovered, which bound to HepG2 cells most tightly. Moreover, the availability of large amounts of well-folded and stable preS1-120 enables us to carry out further structural determination and mechanistic study on HBV infection.     

Plant expression,lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation

Current immunisation programmes against hepatitis B virus (HBV) increasingly often involve novel tri-component vaccines containing—together with the small (S-HBsAg)—also medium and large surface antigens of HBV (M- and L-HBsAg). Plants producing all HBsAg proteins can be a source of components for a potential oral ‘triple’ anti-HBV vaccine. The objective of the presented research was to study the potential of M/L-HBsAg expression in leaf tissue and conditions of its processing for a prototype oral vaccine. Tobacco and lettuce carrying M- or L-HBsAg genes and resistant to the herbicide glufosinate were engineered and integration of the transgenes was verified by PCR and Southern hybridizations. M- and L-HBsAg expression was confirmed by Western blot and assayed by ELISA at the level of micrograms per g of fresh weight. The antigens displayed a common S domain and characteristic domains preS2 and preS1 and were assembled into virus-like particles (VLPs). Leaf tissues containing M- and L-HBsAg were lyophilised to produce a starting material of an orally administered vaccine formula. The antigens were distinctly sensitive to freeze-drying conditions and storage temperature, in the aspect of stability of S and preS domains and formation of multimeric particles. Efficiency of lyophilisation and storage depended also on the initial antigen content in plant tissue, yet M-HBsAg appeared to be approximately 1.5–2 times more stable than L-HBsAg. The results of the study provide indications concerning the preparation of two other constituents, next to S-HBsAg, for a plant-derived prototype oral tri-component vaccine against hepatitis B.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

Detection of hepatitis B virus DNA by polymerase chain reaction in HBsAG negative Senegalese patients suffering from cirrhosis or primary liver cancer

The polymerase chain reaction was used to search for hepatitis B virus (HBV)-DNA sequences in the sera of HBsAg-negative Senegalese patients suffering from liver cirrhosis or liver cancer. Amplified HBV-DNA sequences were detected by hybridization with a digoxigenin-labelled HBV-DNA probe. HBV-DNA was detected in 17% of HBsAg negative Senegalese subjects from the general population and in 44% and 58% of the patients suffering from cirrhosis or primary hepatocellular carcinoma (PHCC) respectively. In the control group, amplified HBV-DNA was detected in 25% of the subjects without HBsAg and anti-HBs antibodies, and in 6% of subjects positive for anti-HBs antibodies. This study confirmed the hypothesis that there is an etiologic link between HBV and PHCC in HBsAg-negative patients.     

TIMM29 interacts with hepatitis B virus preS1 to modulate the HBV life cycle

Hepatitis B virus (HBV), a major global health problem, can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinomas in chronically infected patients. However, before HBV infection can be adequately controlled, many mysteries about the HBV life cycle must be solved. In this study, TIMM29, an inner mitochondrial membrane protein, was identified as an interaction partner of the preS1 region of the HBV large S protein. The interaction was verified by both an immunoprecipitation with preS1 peptides and a GST-pulldown assay. Immunofluorescence studies also showed colocalization of preS1 and TIMM29. Moreover, it was determined that the preS1 bound with amino acids 92–189 of the TIMM29 protein. Infection of HBV in TIMM29-overexpressing NTCP/G2 cells resulted in a significant decrease of HBeAg and both extracellular particle-associated and core particle-associated HBV DNA without affecting cccDNA formation. Comparable results were obtained with TIMM29-overexpressing HB611 cells, which constitutively produce HBV. In contrast, knockout of TIMM29 in NTCP/G2 cells led to a higher production of HBV including HBeAg expression, as did knockout of TIMM29 in HB611. Collectively, these results suggested that TIMM29 interacts with the preS1 region of the HBV large S protein and modulates HBV amplification.     

Prevalence of occult hepatitis B virus infection in kidney transplant recipients

In this cross-sectional study, 207 hepatitis B surface antigen (HBsAg)-negative kidney transplant recipients were evaluated based on demographic and epidemiological data and on the levels of serological markers of hepatitis B virus (HBV) and hepatitis C virus infection and liver enzymes. Patients with HBV or human immunodeficiency virus infection were excluded. Sera were analysed for the presence of HBV-DNA. HBV-DNA was detected in two patients (1%), indicating occult hepatitis B (OHB) infection (the HBV-DNA loads were 3.1 and 3.5 IU/mL in these patients). The results of the liver function tests were normal and no serological markers indicative of HBV infection were detected. The prevalence of OHB infection was low among kidney transplant recipients, most likely due to the low HBsAg endemicity in the general population of the study area.     

毕赤酵母表达的含前S1、前S2和S表位乙肝表面抗原疫苗的制备和免疫原性研究

整合了乙肝表面抗原嵌合基因SS1和SS2的毕赤酵母工程菌株GS115-SS1S2经高密度发酵培养,甲醇诱导,抗原表达量达到300~600mg/L发酵液。SS1S2抗原经细胞破碎、硅胶吸附、疏水层析和凝胶过滤纯化,纯度达99%以上,每升培养物可收获纯化抗原82mg。纯化的SS1S2抗原经Al(OH)3吸附,在NIH小鼠中进行免疫效果评价。三组NIH雌性小鼠,分别腹腔接种2.5μg、0.625μg和0.156μgSS1S2疫苗或商品化的单S疫苗。部分小鼠在30天时采血,测定各疫苗组的ED50值。在SS1S2疫苗组,前S1、前S2和S抗原的ED50值分别为0.46、0.29和0.84μg,而S疫苗组S抗原的ED50为0.99μg。另一部分小鼠分别在7天和14天时采血,考察抗体阳转率与时间的关系。SS1S2疫苗前S1、前S2抗体阳转率在7d和14d时比S抗体的阳转率为高,提示前S抗体出现的时间较早。上述结果显示SS1S2疫苗比单S疫苗具有更好的免疫原性。     

Synthesis of peptides of the preS1-region of the viral hepatitis B envelope protein and localization of antigenic determinants]

We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides.