A synthetic peptide analog of in silico-predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits

Development of a serotyping-capable dengue detection test is hampered by the absence of an identified unique marker that can detect specific dengue virus (DENV) serotype. In the current commercially available antibody-capture diagnostic methods, immobilized nonstructural 1 (NS1) antigen indiscriminately binds and detects immunoglobulin M or immunoglobulin G against any serotype, thus limiting its capability to distinguish existing serotypes of dengue. Identification of dengue serotype is important because certain serotypes are associated with severe forms of dengue as well as dengue hemorrhagic fever. In this study, we aimed to identify an immunogenic epitope unique to DENV2 NS1 antigen and determine the binding specificity of its synthetic peptide mimotope to antibodies raised in animal models. Selection of a putative B-cell epitope from the reported DENV2 NS1 antigen was done using Kolaskar and Tongaonkar Antigenicity prediction, Emini surface accessibility prediction, and Parker hydrophilicity prediction available at the immune epitope database and analysis resource. Uniqueness of the B-cell epitope to DENV2 was analyzed by BLASTp. Immunogenicity of the synthetic peptide analog of the predicted immunogenic epitope was tested in rabbits. The binding specificity of the antibodies raised in animals and the synthetic peptide mimotope was tested by indirect ELISA. A synthetic peptide analog comprising the unique epitope of DENV2 located at the 170th–183rd position of DENV2 NS1 was found to be immunogenic in animal models. The antipeptide antibody produced in rabbits showed specific binding to the synthetic peptide mimotope of the predicted unique DENV2 NS1 immunogenic epitope.     

SOX6 基因3'UTR 区野生型和突变型重组质粒的构建与鉴定

目的:构建含单核苷酸多态性(SNP)位点rs1065024的SOX6基因3'UTR双荧光素酶报告基因载体,并用生物信息学软件预测与rs1065024位点区域相结合的mi RNA,为进一步研究此SNP位点的功能及mi RNA与SOX6基因3'UTR区之间的关系奠定基础。方法:提取人全血基因组DNA,以基因组DNA为模板,通过PCR扩增含SNP位点在内的SOX6基因3'UTR片段,经过胶回收纯化后,将回收的目的片段插入双荧光素酶报告基因载体p MIR-REPORT中,再经DH5a转化扩增,挑单克隆进行菌落PCR并进行质粒提取,对质粒进行双酶切鉴定,最后进行DNA测序鉴定。针对SNP进行定点突变,构建出野生型和突变型重组质粒,并用生物信息学软件预测出与SNP位点相结合的mi RNA。结果:经单菌落质粒测序验证显示带有T碱基的SOX6基因3'UTR重组质粒p MIR-REPORT-3'UTR-T构建成功;经定点突变,成功将p MIR-REPORT-3'UTR-T质粒转变为p MIR-REPORT-3'UTR-C,经比对未引入任何其他突变;生物信息学预测显示,rs1065024位点位于mi R-190b、mi R-190a-5p、mi R-451b、mi R-4791与SOX6基因3'UTR的结合区域,其多态的改变可以影响mi RNA与m RNA的结合效率。结论:本研究成功构建了含SNP位点rs1065024的p MIR-REPORT-SOX6-3'UTR野生型和突变型重组质粒,为今后SOX6基因3'UTR的SNP位点的功能及mi RNA与SOX6基因3'UTR区之间的关系研究奠定基础。     

Genetic variation in the 5' end and NS5B regions of classical swine fever virus genome among Japanese isolates.

Sixteen clinical strains of classical swine fever virus (CSFV) isolated in Japan were subjected to analyses of nucleotide sequence variations in the 5' end and NS5B regions of the genome. These isolates were divided into three genovars, CSFV-1, CSFV-2 and CSFV-3, based on palindromic nucleotide substitutions at the three variable loci in the 5' untranslated region (UTR). Phylogenetic trees constructed from nucleotide sequences in the 5'-UTR and NS5B gene indicated that the CSFV strains were divided into three clusters, I, II and III. CSFV strains included in clusters I, II and III were identical to those in the CSFV-1, CSFV-2 and CSFV-3 genovars, respectively.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines.     

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

Escape from a dominant HLA-B*15-restricted CD8+ T cell response against hepatitis C virus requires compensatory mutations outside the epitope

Antiviral CD8(+) T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8(+) T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8(+) epitopes in NS5B (LLRHHNMVY(2450-2458) and SQRQKKVTF(2466-2474)), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.     

Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies

Background

Epitope-mapping of infectious agents is essential for pathogenesis studies. Since polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) are always polyspecific and can react with multiple epitopes, it is important to distinguish between specific and non-specific reactions. Relative antibody discriminating specificity (RADS) values, obtained from their relative ELISA reactions with L-amino acid peptides prepared in the natural versus reverse orientations (x-fold absorbance natural/absorbance reverse = RADS value) may be valuable for this purpose. PAbs generated against the dengue type-2 virus (DENV-2) nonstructural-1 (NS1) glycoprotein candidate vaccine also reacted with both DENV envelope (E) glycoproteins and blood-clotting proteins. New xKGSx/xSGKx amino acid motifs were identified on DENV-2 glycoproteins, HIV-1 gp41 and factor IXa. Their potential roles in DENV and HIV-1 antibody-enhanced replication (AER) and auto-immunity were assessed. In this study, a) RADS values were determined for MAbs and PAbs, generated in congeneic (H2: class II) mice against DENV NS1 glycoprotein epitopes, to account for their cross-reaction patterns, and b) MAb 1G5.3 reactions with xKGSx/xSGKx motifs present in the DENV-4 NS1, E and HIV-1 glycoproteins and factor IXa were assessed after the introduction of amino acid substitutions, deletions, or intra-/inter-cysteine (C-C) bridges.

Results

MAbs 1H7.4, 5H4.3, 3D1.4 and 1G5.3 had high (4.23- to 16.83-fold) RADS values against single epitopes on the DENV-2 NS1 glycoprotein, and MAb 3D1.4 defined the DENV complex-conserved LX1 epitope. In contrast, MAbs 1G5.4-A1-C3 and 1C6.3 had low (0.47- to 1.67-fold) RADS values against multiple epitopes. PAb DENV complex-reactions occurred through moderately-high (2.77- and 3.11-fold) RADS values against the LX1 epitope. MAb 1G5.3 reacted with xSGKx motifs present in DENV-4 NS1 and E glycoproteins, HIV-1 gp41 and factor IXa, while natural C-C bridge formations or certain amino acid substitutions increased its binding activity.

Conclusions

These results: i) were readily obtained using a standard 96-well ELISA format, ii) showed the LX1 epitope to be the immuno-dominant DENV complex determinant in the NS1 glycoprotein, iii) supported an antigenic co-evolution of the DENV NS1 and E glycoproteins, and iv) identified methods that made it possible to determine the role of anti-DENV PAb reactions in viral pathogenesis.     

Structural lability in stem-loop 1 drives a 5' UTR-3' UTR interaction in coronavirus replication

The leader RNA of the 5′ untranslated region (UTR) of coronaviral genomes contains two stem-loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a noncanonical base pair near A35. Viruses containing destabilizing SL1-ΔA35 mutations also contain one of two specific mutations in the 3′ UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-ΔA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a “dynamic SL1” model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5′ UTR and the 3′ UTR that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.     

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues ⁵AIDITRK¹¹, ⁷²RDELNVL⁷⁸, ²⁵¹KSKHNRREGY²⁶⁰, ²⁶⁹DENGIVLD²⁷⁶, and ³⁴¹DETTLVRS³⁴⁸. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.     

Discovery of new VEGFR-2 inhibitors based on bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives as anticancer agents and apoptosis inducers

Herein, a new wave of bis([1, 2, 4]triazolo)[4,3-a:3'',4''-c]quinoxaline derivatives have been successfully designed and synthesised. The synthesised derivatives were biologically investigated for their cytotoxic activities against HepG2 and MCF-7. Also, the tested compounds were further examined in vitro for their VEGFR-2 inhibitory activity. The most promising derivative 23j was further investigated for its apoptotic behaviour in HepG2 cell lines using flow cytometric and western-plot analyses. Additional in-silico studies were performed to predict how the synthesised compounds can bind to VEGFR-2 and to determine the drug-likeness profiling of these derivatives. The results revealed that compounds 23a, 23i, 23j, 23l, and 23n displayed the highest antiproliferative activities against the two cell lines with IC₅₀ values ranging from 6.4 to 19.4 µM. Furthermore, compounds 23a, 23d, 23h, 23i, 23j, 23l, 23 m, and 23n showed the highest VEGFR-2 inhibitory activities with IC₅₀ values ranging from 3.7 to 11.8 nM, comparing to sorafenib (IC₅₀ = 3.12 nM). Moreover, compound 23j arrested the HepG2 cell growth at the G2/M phase and induced apoptosis by 40.12% compared to the control cells (7.07%). As well, such compound showed a significant increase in the level of caspase-3 (1.36-fold), caspase-9 (2.80-fold), and BAX (1.65-fold), and exhibited a significant decrease in Bcl-2 level (2.63-fold).     

乳房链球菌GapC蛋白的表达及其B细胞抗原表位的预测与鉴定

乳房链球菌Streptococcus uberis的GapC蛋白是一种位于该菌表面的具有甘油醛-3-磷酸脱氢酶活性的蛋白,其参与细胞活动,表现出多种生物学活性,此外还具有良好的抗原性。文中旨在对乳房链球菌GapC蛋白可能的B细胞抗原表位进行预测,分析和验证候选表位肽的免疫原性。利用S. uberis分离株RF5-1克隆gapC基因,构建重组表达质粒pET-28a-GapC,诱导表达GapC重组蛋白,并以纯化蛋白免疫家兔,获得抗GapC多抗。利用生物信息学软件预测并分析GapCB细胞抗原表位的三维结构和空间位置及对GapC蛋白及表位的同源性比较。结果表明,表达纯化了44kDa的GapC蛋白具有良好的反应性。利用表位预测软件筛选并合成针对S.uberisGapC蛋白的6个线性和3个构象优势B细胞表位多肽,三维结构的分析显示,筛选的多肽具有良好的抗原表位形成条件。以纯化的S.uberis GapC蛋白免疫家兔制备多抗,通过间接ELISA对抗原表位进行鉴定。ELISA检测结果显示,9条抗原表位肽均可不同程度地与抗GapC多抗反应,其中表位266AANDSYGYTEDPIVSSD282与多抗反应...     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

Chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in vero cells or human liver cells and attenuated in mice

A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2ADelta30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2ADelta30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Delta30) in the 3' untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2ADelta30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n = 13) or only in HuH-7 cells (n = 7). Each of the 20 ts mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5' and 3' UTRs, with more than one change occurring, in general, per mutant virus. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Delta30 deletion, thereby creating rDEN4Delta30-4995, a recombinant virus which is ts and more attenuated than rDEN4Delta30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.     

Neuroadapted yellow fever virus 17D: genetic and biological characterization of a highly mouse-neurovirulent virus and its infectious molecular clone

A neuroadapted strain of yellow fever virus (YFV) 17D derived from a multiply mouse brain-passaged virus (Porterfield YF17D) was additionally passaged in SCID and normal mice. The virulence properties of this virus (SPYF) could be distinguished from nonneuroadapted virus (YF5.2iv, 17D infectious clone) by decreased average survival time in SCID mice after peripheral inoculation, decreased average survival time in normal adult mice after intracerebral inoculation, and occurrence of neuroinvasiveness in normal mice. SPYF exhibited more efficient growth in peripheral tissues of SCID mice than YF5.2iv, resulting in a more rapid accumulation of virus burden, but with low-titer viremia, at the time of fatal encephalitis. In cell culture, SPYF was less efficient in replication than YF5.2iv in all cell lines tested. The complete nucleotide sequence of SPYF revealed 29 nucleotide substitutions relative to YF5.2iv, and these were distributed throughout the genome. There were a total of 13 predicted amino acid substitutions, some of which correspond to known differences among the Asibi, French viscerotropic virus, French neurotropic vaccine, and YF17D vaccine strains. The envelope (E) protein contained five substitutions, within all three functional domains. Substitutions were also present in regions encoding the NS1, NS2A, NS4A, and NS5 proteins and in the 3' untranslated region (UTR). Construction of YFV harboring all of the identified coding nucleotide substitutions and those in the 3' UTR yielded a virus whose cell culture and pathogenic properties, particularly neurovirulence and neuroinvasiveness for SCID mice, generally resembled those of the original SPYF isolate. These findings implicate the E protein and possibly other regions of the genome as virulence determinants during pathogenesis of neuroadapted YF17D virus in mice. The determinants affect replication efficiency in both neural and extraneural tissues of the mouse and confer some limited host-range differences in cultured cells of nonmurine origin.     

Reactivity of German Cockroach Allergen, Bla g 2, Peptide Fragments to IgE Antibodies in Patients' Sera

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients'' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Epitope mapping of antigenic determinants of hepatitis C virus proteins by phage display

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.