Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.     

Quantitative temporal-spatial distribution of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in post-mortem tissues

Few post-mortem studies have been performed on patients who have died from severe acute respiratory syndrome (SARS). No studies have examined how the SARS-associated coronavirus (SARS-CoV) loads in different organs with respect to time, post-mortem. The aim of this study was to determine the quantitative temporal-spatial distribution of SARS-CoV in the post-mortem tissue samples of seven patients. Quantitation of a house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was undertaken to standardize the amount of tissue tested. SARS-CoV viral load and SARS-CoV/GAPDH RNA ratio for each organ type were related to four time durations: onset of illness to death, death to post-mortem tissue sampling, and total durations of treatment with ribavirin and hydrocortisone. The SARS-CoV/GAPDH RNA ratio remained relatively stable in most organ tissue types for all these time durations. The ratio reached the highest value of equal to or greater than one for lung and small bowel, whereas those for heart, liver, spleen, and kidney were always less than one. It is concluded that SARS-CoV viral loads in these organs remain relatively stable, post-mortem. This quantitative assessment further supports SARS-CoV has a specific tropism for the human respiratory and gastrointestinal tracts, which may be related to the density of SARS-CoV receptors.     

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002–2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the CoV S2 subunit that may preferentially associate with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-CoV and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pre-transmembrane domain of the S2 subunit inhibited SARS-CoV plaque formation by 40–70% at concentrations of 15–30 μM. Interestingly, peptides analogous to the SARS-CoV or MHV loop region inhibited viral plaque formation by >80% at similar concentrations. The observed effects were dose-dependent (IC50 values of 2–4 μM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the CoV peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.     

乙型肝炎核酸疫苗NV—HB／s肌注小鼠后HBsAg的表达和抗 …

目的 研究乙型肝炎核酸疫苗肌注小鼠后在体内的表达及小鼠体液免疫应答的规律,同时观察接种局部组织的病理变化。方法 以乙型肝炎核酸疫苗ＮＶ－ＨＢ／ｓ肌注小鼠,然后定期分批处死,采集肌注局部组织检测ＨＢｓＡｇ（ＡＢＣ免疫组化法）,采集外周血标本检测ＨＢｓＡｇ和抗－ＨＢｓ（ＥＬＩＳＡ法０,并常规病理检查。     

Induction of protective immunity against severe acute respiratory syndrome coronavirus (SARS-CoV) infection using highly attenuated recombinant vaccinia virus DIs

SARS-coronavirus (SARS-CoV) has recently been identified as the causative agent of SARS. We constructed a series of recombinant DIs (rDIs), a highly attenuated vaccinia strain, expressing a gene encoding four structural proteins (E, M, N and S) of SARS-CoV individually or simultaneously. These rDIs elicited SARS-CoV-specific serum IgG antibody and T-cell responses in vaccinated mice following intranasal or subcutaneous administration. Mice that were subcutaneously vaccinated with rDIs expressing S protein with or without other structural proteins induced a high level of serum neutralizing IgG antibodies and demonstrated marked protective immunity against SARS-CoV challenge in the absence of a mucosal IgA response. These results indicate that the potent immune response elicited by subcutaneous injection of rDIs containing S is able to control mucosal infection by SARS-CoV. Thus, replication-deficient DIs constructs hold promise for the development of a safe and potent SARS vaccine.     

严重急性呼吸综合征冠状病毒IgG抗体动态变化的初步观察

目的：探讨严重急性呼吸综合征(Severe Acute Respiratory Syndrome，SARS)冠状病毒抗体IgG的变化规律。方法：采用间接酶联免疫(ELISA)法，对534例发病后1—153天的SARS患者的血清特异性抗体IgG进行检测，并设置对照组进行比较。结果：SARS患者血清IgG抗体阳性率为58．1％，对照组为0．5％，两者具有显著性差异(P=0．000)。SARS患者中，临床症状出现后1—10天、11—30天、31—60天、61—90天、91—120天、≥121天组冠状病毒抗体IgG的阳性率分别为18．8％(3/16例)、63．8％(37／58例)、55．6％(30／54例)、51．4％(71/138例)、62．1％(159／256例)、83．3％(10／12例)。结论：SARS患者发病初期IgG抗体阳性率低，第11天后迅速上升，第4、5个月时持续存在，但持续多长时间尚需进一步观察。     

Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS

The SARS-CoV spike glycoprotein (S) is the main target of the protective immune response in humans and animal models of SARS. Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). The presence of both splice sites and WPRE markedly improved the immunogenicity of S-based DNA vaccines against SARS. Upon immunization of mice with low doses (2 microg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. Our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic RNA viruses.     

False-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid-based western blot assay were rectified by the use of two subunits (S1 and S2) of spike for detection of antibody to SARS-CoV

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.     

A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), can be expressed in Human Embryonic Kidney 293 cells (HEK 293T) and whether such a construct can be used as a DNA vaccine in asthma therapy. The eukaryotic expression vector pcDNA3.1 was used to express the R8 molecule in HEK 293T cells and successful expression of R8 was confirmed using a fluorescence microscope and western blot analysis. The efficacy of R8 as DNA vaccine was also assessed in a mouse asthma model. The in vivo data showed that R8 rectified the TH1/TH2 imbalance typical of allergic inflammation and stimulated the proliferation of regulatory T (Treg) cells. Immunization with the R8 construct also decreased serum allergen-specific IgE production in this mouse asthma model. Our findings suggest that R8 may be a feasible potential DNA vaccine for specific immunotherapy (SIT) in the treatment of allergic asthma.     

MUC1基因疫苗诱导小鼠特异性CTL和体液免疫应答

目的 :观察MUC1基因疫苗诱导小鼠特异性杀伤性T细胞及体液免疫应答的作用。方法 :采用股四头肌肌肉注射 ,将构建的MUC1基因疫苗pcDNA3.1 MUC1免疫雌性BALB/c小鼠 ,每次间隔 3wk ,共 3次。最后 1次免疫后第 3周 ,接种表达MUC1的EMT6乳腺癌细胞进行免疫保护实验。用 4h51Cr释放法检测小鼠脾细胞特异性CTL杀伤活性 ;免疫组化染色法检测小鼠血清特异性抗体的水平。结果 :在效靶比为 10 0∶1、5 0∶1、2 5∶1、12 .5∶1时 ,MUC1基因疫苗免疫组特异性CTL对EMT6靶细胞杀伤活性分别为 5 4 .1%、39.8%、2 6 .4 %和2 0 .1% ,对照组分别为 13.2 %、10 .0 %、8.2 %、7.2 %和 11.7%、9.8%、7.7%、7.0 % ,前者与后二者差异显著 (P <0 .0 1)。免疫组化染色检测显示 ,人乳腺癌组织MUC1呈染色阳性 ;MUC1基因疫苗免疫组仅见 4 0 % (4/ 10 )的小鼠有肿瘤形成 ,而 pcDNA3.1对照组和生理盐水阴性对照组 10 0 %可见肿瘤形成、生长 ,表明MUC1基因疫苗免疫组小鼠具有一定的免疫保护作用。结论 :MUC1基因疫苗可诱导小鼠产生特异性CTL及体液免疫应答 ,对小鼠体内荷瘤可能具有一定的预防作用     

Induction of specific immune responses by severe acute respiratory syndrome coronavirus spike DNA vaccine with or without interleukin-2 immunization using different vaccination routes in mice

DNA vaccines induce humoral and cellular immune responses in animal models and humans. To analyze the immunogenicity of the severe acute respiratory syndrome (SARS) coronavirus (CoV), SARS-CoV, spike DNA vaccine and the immunoregulatory activity of interleukin-2 (IL-2), DNA vaccine plasmids pcDNA-S and pcDNA-IL-2 were constructed and inoculated into BALB/c mice with or without pcDNA-IL-2 by using three different immunization routes (the intramuscular route, electroporation, or the oral route with live attenuated Salmonella enterica serovar Typhimurium). The cellular and humoral immune responses were assessed by enzyme-linked immunosorbent assays, lymphocyte proliferation assays, enzyme-linked immunospot assays, and fluorescence-activated cell sorter analyses. The results showed that specific humoral and cellular immunities could be induced in mice by inoculating them with SARS-CoV spike DNA vaccine alone or by coinoculation with IL-2-expressing plasmids. In addition, the immune response levels in the coinoculation groups were significantly higher than those in groups receiving the spike DNA vaccine alone. The comparison between the three vaccination routes indicated that oral vaccination evoked a vigorous T-cell response and a weak response predominantly with subclass immunoglobulin G2a (IgG2a) antibody. However, intramuscular immunization evoked a vigorous antibody response and a weak T-cell response, and vaccination by electroporation evoked a vigorous response with a predominant subclass IgG1 antibody response and a moderate T-cell response. Our findings show that the spike DNA vaccine has good immunogenicity and can induce specific humoral and cellular immunities in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances the humoral and cellular immune responses. Different vaccination routes also evoke distinct immune responses. This study provides basic information for the design of DNA vaccines against SARS-CoV.     

Type IVB pilus operon promoter controlling expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid gene in Salmonella enterica Serovar Typhi elicits full immune response by intranasal vaccination

Attenuated Salmonella enterica serovar Typhi strains have been considered to be attractive as potential live oral delivery vector vaccines because of their ability to elicit the full array of immune responses in humans. In this study, we constructed an attenuated S. enterica serovar Typhi strain stably expressing conserved nucleocapsid (N) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) by integrating the N gene into the pilV gene, which was under the control of the type IVB pilus operon promoter in S. enterica serovar Typhi. BALB/c mice were immunized with this recombinant strain through different routes: intranasally, orogastrically, intraperitoneally, and intravenously. Results showed that the intranasal route caused the highest production of specific immunoglobulin G (IgG), IgG2a, and secretory IgA, where IgG2a was imprinted as a Th1 cell bias. Moreover, this recombinant live vaccine induced significantly high levels of specific cytotoxic T-lymphocyte activities and increased gamma interferon-producing T cells compared with the parental strain. Our work provides insights into how the type IVB pilus operon promoter controlling SARS-CoV N gene expression in Salmonella might be attractive for a live-vector vaccine against SRAS-CoV infection, for it could induce mucosal, humoral, and cellular immune responses. Our work also indicates that the type IVB pilus operon promoter controlling foreign gene expression in Salmonella can elicit full immune responses by intranasal vaccination.     

DNA vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice

We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.     

板蓝根多糖对猪繁殖与呼吸综合征疫苗免疫猪T细胞亚群的影响

目的：研究板蓝根多糖对猪繁殖与呼吸综合征灭活疫苗免疫猪T细胞亚群及抗体的影响.方法：以板蓝根多糖作为免疫增强剂联合猪繁殖与呼吸综合征灭活疫苗免疫仔猪,通过流式细胞仪检测猪外周血CD3^＋、CD4^＋、CD8^＋细胞百分数,并用ELISA法检测猪血清中抗猪繁殖障碍与呼吸道综合征病毒抗体水平.结果：板蓝根多糖能显著提高仔猪的CD3^＋、CD8^＋淋巴细胞的百分数和特异性抗体滴度.结论：板蓝根多糖能显著增强猪对常规灭活病毒疫苗的免疫应答能力.     

Characterization of the antibody response to a Haemophilus influenzae type b conjugate vaccine in children with recurrent lower respiratory tract infection

Children with recurrent lower respiratory tract infection (RLRI) may respond poorly to polysaccharide antigens. To examine how such children respond to a polysaccharide coupled to a protein carrier, we immunized 15 children with RLRI aged 8–69 months and 15 carefully age-matched healthy controls once with a Haemophilus influenzae type b (Hib) conjugate vaccine. Total IgG subclasses, total antipolysaccharide Hib antibodies, and antipolysaccharide Hib antibodies of IgM, IgG, IgA, and IgG 1–4 specificity were determined by ELISA. There were no significant differences between the two groups in any single total IgG subclass, but total IgG measured as the sum of all four subclasses was significantly lower in the children with RLRI than in the controls ( P = 0.036). Before vaccination, the children with RLRI had significantly less IgG antipolysaccharide Hib antibody than the controls ( P = 0.005), whereas 1 month later they had significantly more IgM antibody (P = 0.038). No other significant differences were found between the groups before or after immunization with respect to antipolysaccharide Hib antibodies. Since naturally occurring IgG antibodies are thought to be aquired partly as a consequence of antigenic stimulation on mucosal surfaces, we hypothesize that the low level of specific IgG found before immunization, as well as the low total IgG in the children with RLRI, may reflect an impaired ability to prime through mucosal surfaces. This is supported by our finding of an increased IgM response to Hib conjugate vaccine in these children, since this isotype predominates in the primary immune response, i.e., in the absence of immunologic memory. In conclusion, children with RLRI can be protected against invasive Hib infection as well as healthy children, but may have an immunodeficiency characterized by defective ability to respond to antigenic stimulation on mucosal surfaces.     

乙型肝炎病毒DNA疫苗电转加强含S-PreS1颗粒的蛋白疫苗在小鼠中免疫效果的研究

目的探索新型有效的HBV治疗性疫苗研制的策略。方法构建含S与PreS1融合基因的HBVDNA疫苗,采用两次蛋白疫苗（含不同佐剂）肌内注射初免,一次DNA疫苗皮内电转免疫加强的联合免疫方式,在小鼠中分析比较了各疫苗所引起的体液与细胞免疫应答。结果HBVDNA疫苗皮内注射加电转可明显增强表面抗原（s）特异的细胞免疫应答（IFN-γ ELISpot分析）及PreS1特异性抗体水平,并以蛋白疫苗加铝佐剂初免组细胞免疫增强效果最明显。结论HBSS1 DNA疫苗结合皮内注射＋电转方式可明显加强含S-PreS1颗粒的蛋白疫苗在小鼠中免疫效果,明显高于蛋白疫苗单独应用。这些研究结果为新型HBV治疗性疫苗的研制与应用提供了依据。     

The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.     

表达丙型肝炎病毒NS3和Core融合蛋白基因的DNA疫苗免疫方案优化

目的 本研究旨在构建一种包含丙型肝炎病毒(HCV)保守区基因的新型DNA疫苗,并在小鼠模型中使用电转技术优化其免疫原性.方法 首先,我们构建了包含HCV非结构蛋白NS3和核心蛋白Core部分基因序列的DNA疫苗,并证实了其表达;然后采用不同的体内电转方式于第0、4周分别免疫BALB/c小鼠,比较分析不同免疫方案的体液免疫(特异性IgG与抗体亚类)与细胞免疫应答(IFN-γ ELISPOT)的效果.结果 使用电转技术可显著增强新型DNA疫苗免疫原性,采用皮内注射加卡钳电极电转的方式产生最强NS3特异性T细胞免疫反应.结论 包含HCV保守区基因的新型DNA疫苗可通过优化电转技术增强免疫应答效果.这为我们下一步优化HCV DNA疫苗的免疫方案提供了依据.     

Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection

Antibodies directed to the HBs antigen indicate viral clearance and the development of life-long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti-HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs-specific antibody response. The existence of anti-HBs-secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self-limited hepatitis B, HBs-specific B cells were demonstrated with a high frequency despite undetectable anti-HBs serum antibodies. HBV-immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti-HBs-secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti-HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs-specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs-specific B cell response in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV-immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti-HBs-producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs-specific B cells years after resolution of HBV infection. In chronic HBV carriers, however, deficient HBs-specific T and B cell responses were observed.