A Dominant Antigenic Region of the Hantaan Virus Nucleocapsid Protein is Located within a Amino-Terminal Short Stretch of Hydrophilic Residues

The nucleocapsid (N) protein of the Hantaan virus (HTNV) is a major viral antigen that induces a strong antibody response during the acute phase of infection. By immunoblot analyses of the recombinant N proteins using human sera of the hemorrhagic fever with renal syndrome (HFRS), we have confirmed previous finding by other investigators of the presence of a highly antigenic region near the amino terminus of the HTNV N protein. We have further located the antigenic region within a short stretch of hydrophilic sequences between the 26 and the 46th amino acid residues. The recombinant glutathione S-transferase fusion proteins containing this region was expressed as a soluble form in a large quantity in Escherichia coli, and purified by a single-step affinity chromatography. The recombinant antigen also showed a similar, but a weaker reactivity with human antisera to Seoul virus (SEOV), the virus most closely related to HTNV.     

Immunization with Toscana virus N-Gc proteins protects mice against virus challenge

Toscana virus (TOSV) is an emerging virus, circulating in the Mediterranean area, that is responsible for aseptic meningitis, meningoencephalitis, and encephalitis. The development of a vaccine that could provide complete protection from TOSV infection is needed. In this study we investigated the capacity of TOSV structural proteins, nucleocapsid protein N and the two Gc and Gn glycoproteins, produced as recombinant proteins, in an animal model. In particular, we investigated their role in inducing specific and protective immune responses against virus infection. Mice were immunized intraperitoneally using TOSV antigens singly or in combination. The results show that only the N-Gc combination was able to protect 100% of animals from a lethal challenge with a neurovirulent strain of TOSV. This potential vaccine induces high serum antibody titres with neutralizing activity and it is safe for animals. Moreover, immunization induces a virus specific cell-mediated immune response, in particular a CD8+ T cell response associated with a marked expression of interferon gamma. These results indicate that the N+Gc viral antigen combination could be useful for future development of a vaccine controlling the spread of this emerging virus that could pose a new threat for humans.     

The humoral response to Puumala virus infection (nephropathia epidemica) investigated by viral protein specific immunoassays

Summary Enzyme-linked immunosorbent assays for determination of antibodies directed to the nucleocapsid protein (N) or to either of the two envelope glycoproteins (G1 and G2) of Puumala virus were designed and evaluated. The assays were proven to be entirely restricted for each viral structural protein by biotin-labelled monoclonal antibodies. Sera from sequentially bled nephropathia epidemica patients (acute, convalescent, and 2-year sera) and sera from 10–20 year convalescents were examined for antibody specificity. All but one (n=19) acute phase sera were shown to contain IgM antibodies directed to all three viral proteins. In the convalescent specimens the proportions of IgM to the different viral components were similar, but lower, when compared to the acute samples. Low levels of IgM against N and G2 were found in two out of ten 2-year sera. No virus-specific IgM were detected in sera drawn 10–20 years after infection. IgG antibodies to all three viral proteins were detected in all except one acute phase serum. The IgG response initially increased more rapidly to N, as compared to the anti-glycoprotein responses. The levels of glycoprotein-specific IgG were considerably increased 2 years after the disease, when compared to the levels detected in the convalescent specimens. The levels and specificities of IgG in very late convalescent sera (drawn 10–20 years after disease) resembled those detected 2 years after infection.     

A soluble and heat-resistant form of hantavirus nucleocapsid protein for the serodiagnosis of HFRS

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne viral zoonosis characterized by fever, hemorrhagic manifestations, and renal disorder. The causative agent of HFRS has been identified as a hantavirus. Hantavirus nucleocapsid proteins have recently been shown to be immunodominant antigens in HFRS, inducing an early and long-lasting immune response, and their amino termini are sensitive tools for the detection of specific antibodies in HFRS patient sera. Previous work has demonstrated that the introduction of the acidic tail of alpha-synuclein (ATS) into heat-labile proteins protects them from heat-induced aggregation. In this study, the ATS peptide was introduced into the N-terminal antigenic portion of the nucleocapsid proteins (amino acid residues 1-70) of the Hantaan virus (HTNV-delta N) and Seoul virus (SEOV-delta N). The recombinant ATS-HTNV-delta N and ATS-SEOV-delta N fusion proteins were heat-resistant, and the proteins purified by heat treatment were immunoreactive to sera from patients with HFRS. Compared with sera from patients with leptospirosis and scrub typhus, sera from patients with HFRS showed much higher reactivity in ATS-HTNV-delta N- or ATS-SEOV-delta N-based IgG ELISAs. Immunoblotting analysis revealed that only sera from patients with HFRS specifically recognized the ATS-HTNV-delta N and ATS-SEOV-delta N, indicating that the ATS-HTNV-delta N and ATS-SEOV-delta N were highly purified species without any other immunoreactive proteins as contaminants. These data demonstrate that the ATS-HTNV-delta N and ATS-SEOV-delta N fusion proteins offer a safe and inexpensive source of pure and specific antigen for large-scale diagnosis and seroepidemiological studies of HFRS, and that ATS-fusion technology can also be utilized to solubilize other antigens that could be used for large-scale diagnosis and seroepidemiological studies of other diseases.     

狂犬病病毒糖蛋白和核蛋白基因的克隆及其重组蛋白在昆虫细胞中的表达

目的从国内狂犬病疫苗ａＧ株中克隆狂犬病病毒糖蛋白（ＧＰ）基因和核蛋白（ＮＰ）基因，应用杆状病毒－昆虫细胞表达系统使其在昆虫细胞中表达。方法从狂犬病病毒感染细胞上清液中提取病毒ＲＮＡ，应用ＲＴ－ＰＣＲ方法扩增ＧＰ基因和ＮＰ基因。扩增的基因与转移质粒连接并转化大肠杆菌，得到重组转移质粒。将其与野生杆状病毒（ＡｃＭＮＰＶ）线性ＤＮＡ共转染Ｓｆ９昆虫细胞，通过有限稀释法筛选含有ＧＰ基因和ＮＰ基因重组病毒。初步检测了重组蛋白的抗原性。结果用ＲＴ－ＰＣＲ方法扩增得到ＧＰ基因和ＮＰ基因，通过重组转移后重组病毒感染的细胞经免疫印染实验表明可分别表达ＧＰ和ＮＰ重组蛋白。重组蛋白的分子量分别为５８×１０３和５３×１０３。用重组病毒感染的细胞免疫小鼠后可诱导动物产生特异性抗体。结论可以应用杆状病毒－昆虫细胞表达系统表达狂犬病病毒ＧＰ和ＮＰ重组蛋白，为开发基因工程化狂犬病疫苗提供有意义的资料     

利用反向遗传学技术构建H5N9重组流感病毒株

目的 利用反向遗传学技术构建来源人感染禽流感病毒H5N1和H7N9 HA和NA基因的H5N9亚型禽流感病毒.方法 全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,插入到pHW2000载体,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒.结果 核酸测序、HA和NA基因转录和表达检测、细胞病变分析确定利用该反向遗传学系统可以成功拯救H5N9病毒.重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法拯救H1N1病毒.结论 利用反向遗传学技术成功构建一株H5N9重组病毒.     

Humoral immune response to rotavirus NSP4 enterotoxin in Spanish children

The rotavirus non-structural protein 4 (NSP4) has been shown to play a crucial role in rotavirus-induced diarrhea, acting as a viral enterotoxin. It has also been demonstrated that antibody to NSP4 can reduce the severity of rotavirus-induced diarrhea in newborn mice. Two recombinant baculoviruses, expressing the NSP4 protein from the SA11 and Wa rotavirus strains, genotypes A and B, respectively, were used to produce and purify these glycoproteins, which were applied as antigen in an enzyme-linked immunosorbent assay (ELISA) to test the specific antibody response to NSP4 in human sera. Serum samples from 30 children convalescing from a rotavirus infection, from 54 healthy children under 5-years-old, and from 49 adults were tested to determine the presence of antibodies to the viral enterotoxin and to rotavirus structural proteins. Seventy percent of the sera from rotavirus-infected children showed an IgG antibody response to either one or both NSP4 proteins used in this study, although the response was weak. However, IgG antibodies towards either one or both NSP4 proteins were only detected in 26% of the non-convalescent healthy children and in only 18% of the adults. No serum IgA antibodies towards NSP4 were found in this study. IgG antibody recognition of the NSP4 protein from the SA11 and Wa rotavirus strains was not always heterotypic.     

Neutralization of Toscana virus is partially mediated by antibodies to the nucleocapsid protein

The envelope glycoproteins G1/G2 of Toscana virus (TOSV) seem to have the most important protective role in stimulating antibodies against the disease in humans, as well as antibodies against the Nucleoprotein (N), a partial neutralizing activity. Mice immunized with TOSV recombinant Nucleoprotein developed a strong humoral response to the TOSV that revealed the presence of neutralizing antibody than in vitro assay. The neutralizing antibody titre of mice immunized with the whole TOSV was analyzed before and after absorption of the sera with the recombinant N protein. A decrease of the neutralizing activity was observed in the treated sera. Similar results were obtained absorbing human anti-TOSV positive sera with the recombinant N protein. This study was designed to identify the nature of antibodies produced against the N protein of TOSV in mice and to establish correlation with antibodies produced in humans by natural infection.     

Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.     

Diagnostic potential of baculovirus-expressed rubella virus envelope proteins.

The envelope glycoproteins E1 and E2 of rubella virus were abundantly expressed in Spodoptera frugiperda Sf9 insect cells by using a baculovirus expression vector. The recombinant protein products were purified by immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The purified recombinant antigen consisted of the envelope polypeptides, corresponding to the viral E1 and E2 proteins, and a polyprotein precursor (molecular mass, 90 to 95 kDa). The antigen was reactive with human convalescent-phase sera in immunoblot analysis, and the reactivity correlated well (r = 0.861) with that of a whole-virus antigen when tested by EIA by using a total of 106 rubella virus immunoglobulin G-positive and -negative serum specimens. When the sera from patients with recent rubella virus infection were tested with the recombinant glycoproteins by EIA, the correlation was not as close (r = 0.690). However, all of the 26 serum specimens were reactive with the recombinant antigen. The results demonstrate that these bioengineered antigens have a potential for use in routine diagnostic assays of rubella virus immunity and recent infection.     

森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及免疫活性测定

目的为了表达森林脑炎病毒prME蛋白,为森林脑炎快速诊断试剂的研制奠定基础。方法经过RTPCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,以杆状病毒昆虫细胞表达系统成功地表达了森林脑炎病毒MDJ01株prME蛋白。结果从感染细胞上清中电镜观察到重组蛋白形成的球型颗粒,说明重组病毒感染细胞后产生病毒样表达颗粒(viruslikeparticlesVLPs),并且分泌至细胞外。免疫印迹试验和间接免疫荧光试验表明,表达的重组蛋白能够与抗森林脑炎病毒抗体特异结合,具有良好的抗原性。ELISA和间接免疫荧光染色证实,重组prME蛋白可以作为抗原用于检测患者血清特异性抗体。结论在昆虫细胞中表达的prME具有良好的抗原性,本研究为森林脑炎快速特异诊断试剂研制奠定了基础。     

False-positive sera do not react with human immunodeficiency virus (HIV) gag-encoded recombinant antigen

Ten sera from healthy blood donors positive by enzyme-linked immunoadsorbent assay (ELISA) were studied by immunoblot assay using natural and recombinant proteins. They interacted only with p17 or p24 proteins but were nonreactive with a recombinant protein (RP 50), which carries antigenic determinants to p17 and p24. Reactions were not blocked by preincubation of sera with genetically engineered p17 and p24 or purified viral p24, indicating that some new epitopes were formed during the Western blot procedure. Recombinant gag-encoded protein is required for confirmation of human immunodeficiency virus (HIV) seropositivity.     

Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.     

Molecular dissection of the human B cell response against cytomegalovirus infection by lambda display

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, is the main cause of congenital abnormalities and mental retardation in newborns and is also responsible for severe life-threatening complications in immunocompromised individuals, including AIDS patients and transplant recipients. The disorders generated by cytomegalovirus are closely associated with the competence of the host immune system and both humoral and cell-mediated mechanisms are involved in the response to viral infection. To identify viral proteins recognized by host antibody responses, a cytomegalovirus genome library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals with congenital or acquired infection allowed the identification of a wide panel of recombinant bacteriophages carrying cytomegalovirus B cell epitopes. Epitope-containing fragments within the families of tegument proteins (pUL25, pUL32), structural proteins (pUL48, pUL56) and glycoproteins (pUL55) were identified. Moreover, library screening permitted isolation of phage clones carrying an antigenic region of an uncharacterized HCMV protein encoded by the UL71 open reading frame (ORF), highlighting the potential of lambda display technology in antigen and epitope discovery.     

乙型肝炎病毒PreS1基因的原核表达及免疫学性质鉴定

目的 构建含乙型肝炎病毒(HBV)PreS1基因的原核表达载体,获得高纯度、有生物活性的PreS1重组蛋白.方法 采用PCR法扩增PreS1及其N末端和C末端部分基因序列,克隆入原核表达载体pGST-MOLUC.重组质粒经IPTG诱导表达后进行SDS-PAGE电泳分析和Western blot检测.用谷胱甘肽-Sepharose 4B凝胶亲和层析纯化的重组融合蛋白免疫新西兰家兔,制备GST-PreS1融合蛋白的抗血清.进一步用ELISA分析融合蛋白特异抑制HBV病毒与抗体结合的能力.结果 重组质粒转化宿主菌后成功诱导出Mr39 000、31 000和32 000的GST-PreS1、GST-PreS1N及GST-PreS1C融合蛋白,均与预期相对分子质量相符.Western blot检测获得特异的杂交条带.采用谷胱甘肽-Sepharose 4B凝胶纯化的融合蛋白纯度约为90%左右.融合蛋白免疫家兔后抗体滴度达到10-7.病毒捕获ELISA实验表明,融合蛋白能特异抑制病毒与家兔免疫血清结合.结论 GST-PreS1融合蛋白能够在大肠杆菌中高效表达,其纯化产物是研究PreS1基因在HBV感染过程中作用的有用工具.     

A Major Antigenic Domain of Hantaviruses is Located on the Aminoproximal Site of the Viral Nucleocapsid Protein

Hantavirus nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response. Recombinant proteins representing various regions of the nucleocapsid proteins as well as segments of the G1 and the G2 glycoproteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E. coli. The antigenicity of these proteins was tested in enzyme immunoassays and immunoblots. These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral nucleocapsid proteins. This fragment was recognized by all HFRS patient sera tested (n=128). The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays. Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site. A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.     

Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins

Autographa californica nuclear polyhedrosis viral recombinants containing coding information for the Rift Valley fever virus (RVFV) envelope glycoproteins (G1 and G2) and varying amounts of preglycoprotein coding sequences were prepared by using transfer vectors pAc373 or pAcYM1. Expression products were processed to yield proteins indistinguishable from authentic G1 and G2 by gel electrophoresis. The immunogenic properties of the expressed proteins were assessed by immunizing mice and challenging with RVFV. A single inoculation with lysates of cells infected with recombinants expressing both G1 and G2 induced neutralizing antibody responses in mice and protected them from an otherwise lethal challenge with RVFV. Lysates of cells infected with a recombinant expressing only G2 also induced a protective response after two immunizations. Survivors displayed elevated antibody titers to G1 and G2 and also developed antibodies to the RVFV nucleocapsid protein, the latter allowing discrimination from vaccinated mice and indicating that animals had survived infection. Nonimmune mice were protected from lethal RVFV infection by passive transfer of sera from animals immunized with recombinant antigens, indicating that a humoral immune response is sufficient to protect against RVFV.     

Soluble recombinant HTLV-1 surface glycoprotein competitively inhibits syncytia formation and viral infection of cells

Efficient entry into, and infection of, human cells by human T-cell leukaemia virus type-1 (HTLV-1) is mediated by the viral envelope glycoproteins, gp46 and gp21. The gp46 surface glycoprotein binds to an as yet unidentified cell surface receptor, thereby, allowing the gp21 transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. In the absence of membrane fusion viral penetration and entry into the host cell cannot occur. The envelope glycoproteins are also a major target for neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and represent ideal constituents for a recombinant HTLV-1 vaccine. Given the importance of the envelope proteins in HTLV-1 pathogenesis there is increasing interest in obtaining sufficient quantities of these proteins for biochemical, biophysical and biological analyses. We have now developed a system for production of large amounts of a glycosylated and functional form of soluble recombinant gp46 (sRgp46), and have used this recombinant material for analysis of envelope function and receptor binding activity. We find that, the sRgp46 molecules expressed in our system are immunologically indistinguishable from the native virally expressed surface glycoproteins; that sRgp46 binds to T-cells in a dose dependent and saturable manner; and that cell surface binding by sRgp46 can be inhibited by neutralising antibodies. Importantly, we demonstrate that these sRgp46 molecules potently inhibit syncytia formation and viral infection of target cells, and that regions outwith the SU domain of envelope are not required for binding to target cells or for inhibiting membrane fusion. The sRgp46 produced in our study will provide new opportunities to investigate envelope-receptor interactions, and will be of utility in defining the conformationally sensitive antigenic determinants of the HTLV-1 surface glycoprotein.     

A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease

Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.