The structural proteins of infectious pancreatic virus are not glycosylated.

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.     

Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.     

Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs

VP7 of group A rotavirus(RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector,three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains.Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6 F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.     

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.     

The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had Mr 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was suggested by antigen binding to peanut lectin. Pulse and chase experiments using [35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5-6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.     

Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry

Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.     

Carbohydrates Facilitate Correct Disulfide Bond Formation and Folding of Rotavirus VP7

It is well established that glycosylation is essential for assembly of enveloped viruses, but no information is yet available as to the function of carbohydrates on the nonenveloped but glycosylated rotavirus. We show that tunicamycin and, more pronouncedly, a combination of tunicamycin and brefeldin A treatment caused misfolding of the luminal VP7 protein, leading to interdisulfide bond aggregation. While formation of VP7 aggregates could be prevented under reducing conditions, they reoccurred in less than 30 min after a shift to an oxidizing milieu. Furthermore, while glycosylated VP7 interacted during maturation with protein disulfide isomerase, nonglycosylated VP7 did not, suggesting that glycosylation is a prerequisite for protein disulfide isomerase interaction. While native NSP4, which does not possess S-S bonds, was not dependent on N-linked glycosylation or on protein disulfide isomerase assistance for maturation, nonglycosylated NSP4 was surprisingly found to interact with protein disulfide isomerase, further suggesting that protein disulfide isomerase can act both as an enzyme and as a chaperone. In conclusion, our data suggest that the major function of carbohydrates on VP7 is to facilitate correct disulfide bond formation and protein folding.     

B cell epitopes within VP1 of type O foot-and-mouth disease virus for detection of viral antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141–160 (epitope1), tandem repeat 200–213 (epitope2 (+2)) and the combination of two epitopes (epitope1–2) was genetically cloned into the prokaryotic expression vector pP_ROExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 (+2) and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 (+2), GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.     

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.     

Antigenic sequences of poliovirus recognized by T cells: serotype-specific epitopes on VP1 and VP3 and cross-reactive epitopes on VP4 defined by using CD4+ T-cell clones.

A panel of poliovirus-specific murine CD4+ T-cell clones has been established from both BALB/c (H-2d) and CBA (H-2k) mice immunized with Sabin vaccine strains of poliovirus serotype 1, 2, or 3. T-cell clones were found to be either serotype specific or cross-reactive between two or all three serotypes. Specificity analysis against purified poliovirus proteins demonstrated that T-cell clones recognized determinants on the surface capsid proteins VP1, VP2, and VP3 and the internal capsid protein VP4. Panels of overlapping synthetic peptides were used to identify eight distinct T-cell epitopes. One type 3-specific T-cell clone recognized an epitope within amino acids 257 and 264 of VP1. Three T-cell epitopes corresponding to residues 14 to 28, 189 to 203, and 196 to 210 were identified on VP3 of poliovirus type 2. The remaining four T-cell epitopes were mapped to an immunodominant region of VP4, encompassed within residues 6 and 35 and recognized by both H-2d and H-2k mice. The epitopes on VP4 were conserved between serotypes, and this may account for the predominantly cross-reactive poliovirus-specific T-cell response observed with polyclonal T-cell populations. In contrast, T-cell clones that recognize epitopes on VP1 or VP3 were largely serotype specific; single or multiple amino acid substitutions were found to be critical for T-cell recognition.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Molecular characterization of a subgroup specificity associated with the rotavirus inner capsid protein VP2

Group A rotaviruses are classified into serotypes, based on the reactivity pattern of neutralizing antibodies to VP4 and VP7, as well as into subgroups (SGs), based on non-neutralizing antibodies directed against VP6. The inner capsid protein (VP2) has also been described as a SG antigen; however, little is known regarding the molecular determinants of VP2 SG specificity. In this study, we characterize VP2 SGs by correlating genetic markers with the immunoreactivity of the SG-specific monoclonal antibody (YO-60). Our results show that VP2 proteins similar in sequence to that of the prototypic human strain Wa are recognized by YO-60, classifying them as VP2 SG-II. In contrast, proteins not bound by YO-60 are similar to those of human strains DS-1 or AU-1 and represent VP2 SG-I. Using a mutagenesis approach, we identified residues that determine recognition by either YO-60 or the group A-specific VP2 monoclonal antibody (6E8). We found that YO-60 binds to a conformationally dependent epitope that includes Wa VP2 residue M328. The epitope for 6E8 is also contingent upon VP2 conformation and resides within a single region of the protein (Wa VP2 residues A440 to T530). Using a high-resolution structure of bovine rotavirus double-layered particles, we predicted these epitopes to be spatially distinct from each other and located on opposite surfaces of VP2. This study reveals the extent of genetic variation among group A rotavirus VP2 proteins and illuminates the molecular basis for a previously described SG specificity associated with the rotavirus inner capsid protein.     

Alkali-catalyzed β-elimination of periodate-oxidized glycans: A novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed -elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.     

Mutations conferring resistance to neutralization with monoclonal antibodies in type 1 poliovirus can be located outside or inside the antibody-binding site.

Antigenic variants resistant to eight neutralizing monoclonal antibodies were selected from wild (Mahoney) and attenuated (Sabin) type 1 infectious poliovirions. Cross-immunoprecipitation revealed interrelationships between epitopes which were not detected by cross-neutralization. Operational analysis of antigenic variants showed that seven of eight neutralization epitopes studied were interrelated. Only one neutralization epitope, named Kc, varied independently from all the others. This latter, recognized by C3 neutralizing monoclonal antibody, was present not only on infectious virions but also on heat-denatured (C-antigenic) particles and on isolated capsid protein VP1. Loss of the neutralization function of an epitope did not necessary result from the loss of its antibody-binding capacity. Such potential, but not functional, neutralization epitopes exist naturally on Mahoney and Sabin 1 viruses. Their antibody-binding property could be disrupted by isolating antigenic variants in the presence of the nonneutralizing monoclonal antibody and anti-mouse immunoglobulin antibodies. Single-point mutations responsible for the acquisition of resistance to neutralization in the antigenic variants were located by sequence analyses of their genomes. Mutants selected in the presence of C3 neutralizing monoclonal antibody always had the mutation located inside the antibody-binding site (residues 93 through 103 of VP1) at the amino acid position 100 of VP1. On the contrary, antigenic variants selected in the presence of neutralizing monoclonal antibodies reacting only with D-antigenic particles had mutations situated in VP3, outside the antibody-binding site (residues 93 through 103 of VP1). The complete conversion of the Mahoney to the Sabin 1 epitope map resulted from a threonine-to-lysine substitution at position 60 of VP3.     

Identification of VP1 peptides diagnostic of encephalomyocarditis virus from swine

Background

Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in China. EMCV VP1 protein was one of the most important structural proteins and played an important role in the protective immunity. In this study, 10 monoclonal antibodies (McAbs) against EMCV VP1 were screened and identified.

Results

Epitope mapping results indicated that McAbs (6E11, 7A7, 7C9) specifically recognized the linear epitopes V(2)ENAEK(7), McAbs (1D1, 2A2, 5A1, 5A11, 5G1) recognized the epitope F(19)VAQPVY(25), and McAbs 1G8 and 3A9 recognized P(42)IGAFTVK(49). Protein sequence alignment of VP1 with 16 EMCV isolates indicated that the epitope F(19)VAQPVY(25) was conserved in all the reference strains. The epitopes P(42)IGAFTVK(49) and V(2)ENAEK(7) only had 1 or 2 variable amino acid among the reference strains. The 3D model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle.

Conclusions

In this study, ten McAbs against EMCV VP1 were developed and three B-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in VP1. All the results herein will promote the future investigations into the function of VP1 of EMCV and development of diagnostic methods of EMCV.

     

Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers.

Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.     

传染性法氏囊病病毒VP3抗原表位的鉴定

利用肽扫描技术对4株IBDV VP3的单克隆抗体(HRB-3F、HRB-7B、HRB-7C和HRB-10E)的抗原表位进行了研究.通过Western blot和ELISA鉴定,将HRB-3F和HRB-7B的抗原表位定位于VP3 109～119 aa(位于IB-DV聚合蛋白的864～874 aa),HRB-7C和HRB-10E的抗原表位定位于VP3 177～190 aa(位于IBDV聚合蛋白的932～945 aa).进一步检测其反应原性及免疫原性,结果表明,这两个表位均能与抗IBDV阳性血清反应.将这两个表位短肽免疫BALB/c小鼠,其血清可以和IBDV反应,具有较好的免疫原性.与D6948、HK46和UK661等多株IBDV相应区域的同源性进行了比较,结果显示,这两个表位在多种毒株中同源性为100%.通过IBDV VP3抗原表位的研究,筛出两个新的保守线性表位并进行精确定位,对进一步分析IBDV结构与功能以及建立以表位为基础的抗原抗体诊断方法具有重要的意义.     

含禽流感病毒M2e 氨基端抗原表位的重组传染性法氏囊病毒VP2 蛋白的免疫原性鉴定

【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。     

The VP7 genes of two G9 rotaviruses isolated in 1980 from diarrheal stool samples collected in Washington, DC, are unique molecularly and serotypically

In a retrospective study of archival diarrheal stool samples collected from 1974 to 1991 at Children's Hospital National Medical Center, Washington, DC, we detected three genotype G9P[8] viruses in specimens collected in 1980, which represented the earliest human G9 viruses ever isolated. The VP7 genes of two culture-adapted 1980 G9 viruses were phylogenetically related closely to the lineage 2 G9 virus VP7 gene. Unexpectedly, however, the VP7s of the 1980 G9 viruses were more closely related serotypically to lineage 3 VP7s than to lineage 2 VP7, which may be supported by amino acid sequence analyses of the VP7 proteins.     

Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.