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.     

Humoral immune response to functional regions of human cytomegalovirus glycoprotein B

Sera from patients with primary human cytomegalovirus (HCMV) infections, both acute and convalescent phase, and from HCMV-seropositive healthy subjects were analyzed to determine whether the sera would recognize antigenic domains on HCMV glycoprotein B (gB) that function in virion infectivity and spread of virus from cell to cell. The intact gB molecule, amino-terminal derivatives of different lengths, and internal deletion derivatives were expressed in eukaryotic cells and reacted by immunofluorescence with the sera. All convalescent-phase sera and most sera from healthy seropositive individuals reacted with full-length gB and with an amino-terminal derivative containing 687 amino acids (aa), gB-(1–687); approximately half of the sera recognized an amino-terminal derivative of 447 aa, gB-(1–447), and one-third reacted with the shortest deletion derivative of 258 aa, gB-(1–258). Of the acute-phase sera, 77% recognized intact gB and gB-(1–687), 32% recognized gB-(1–447), and 14% recognized gB-(1–258). Deletion of aa 548 to 618 dramatically reduced the percentage of reactive sera, whereas deletion of aa 411 to 447 had a minor impact on reactivity of sera. To investigate the epitope specificity of human antibodies to gB, we carried out competition experiments between human sera with neutralizing activity and selected monoclonal antibodies (mAbs) to conformational epitopes on gB. We found that antibodies in human sera preclude syncytium formation in UB15-11 glioblastoma cells constitutively expressing gB and compete with certain murine mAbs that block virus entry into cells and transmission of infection from cell to cell. Our results show that HCMV-immune human sera contain antibodies to functional regions on gB, and the abundance of these antibodies in convalescent-phase sera suggests that they may play a central role in limiting dissemination of virus in the host. J. Med. Virol. 52:451–459, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides

The human nuclear autoantigenic sperm protein, NASP, is a testicular histone-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In this study to define the boundaries of antigenic regions and epitope recognition pattern, recombinant deletion mutants spanning the entire protein coding sequence and a human NASP cDNA sublibrary were screened with vasectomy patients' sera. Employing panel sera from 21 vasectomy patients with anti-sperm antibodies, a heterogeneous pattern of autoantibody binding to the recombinant polypeptides was detected in ELISA and immunoblotting. The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Antigenic sites preferentially recognized by the individual patients' sera were located within aa 32-352 and aa 572-787. Using a patient's serum selected for its reactivity to the whole recombinant protein in Western blots, cDNA clones positive for the C-terminal domain of the molecule were identified. The number and location of linear epitopes in this region were determined by synthetic peptide mapping and inhibition studies. The epitope-containing segment was delimited to the sequence aa 619-692 and analysis of a series of 74 concurrent overlapping 9mer synthetic peptides encompassing this region revealed four linear epitopes: amino acid residues IREKIEDAK (aa 648-656), KESQRSGNV (aa 656-664), AELALKATL (aa 665-673) and GFTPGGGGS (aa 680-688). All individual patients' sera reacted with epitopes within the sequence IREellipsis.GGS (aa 648-688). The strongest reactivity was displayed by peptides corresponding to the sequence AELALKATL (aa 665-673). Thus, multiple continuous autoimmune epitopes in NASP involving sequences in the conserved C-terminal domain as well as in the less conserved testis-specific N-terminal region comprising the histone-binding sites, as predicted for an antigen-driven immune response, may be a target of autoantibodies in vasectomized men and may provide a relevant laboratory variable to describe more accurately the spectrum of autoantibody specificities associated with the clinical manifestation of vasectomy.     

Identification of B cell epitopes at the C-terminus of latency-associated nuclear protein of the kaposi's sarcoma-associated herpesvirus

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays a key role in the induction of cell transformation, maintenance of viral episome, and modulation of immune response in human. To identify the presence of B cell epitopes within C-terminus of LANA and to characterize the monoclonal antibodies (MAbs) against this protein, we expressed the C-terminal region at aa 794-1000 of LANA (pLANA-C) in Escherichia coli as a fusion protein. KSHV-positive human sera were able to recognize the recombinant LANA-C in the Western blot analysis and ELISA. Mapping of antigenic epitopes of pLANA-C by KSHV-positive human sera revealed two B cell antigenic epitopes located at aa 846-854 and aa 794-822. The MAb 3F11 recognized a region between at aa 840 to 846 of LANA and exhibited a strong and specific binding to both pLANA-C and native viral LANA. These findings showed that pLANA-C and MAb 3F11 could be used for the detection of KSHV antibodies in human sera and for the advanced study of biological functions of LANA.     

Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination.     

Linear epitopes of two different autoantigens-La/SSB and myelin basic protein--with a high degree of molecular similarity,cause different humoral immune responses

The region 147-154aa of La/SSB presents 83% sequence similarity with the 139-146aa region of the human myelin basic protein (MBP). The aim of this study was to investigate the prevalence and significance of antibodies against both epitopes in sera from patients with systemic autoimmune diseases, and to compare the humoral responses produced after rabbit immunization. Peptides 147-154aa of La/SSB and 139-146aa of the MBP were attached on tetrameric sequential oligopeptide carriers and used for immunizations of New Zealand White rabbits. Antibodies to immunizing peptides, as well as to the peptides corresponding to other previously defined La/SSB epitopes (289-308aa, 349-364aa), to the intact human MBP (hMBP) and to the recombinant human La/SSB (rechLa) proteins, were identified using specific ELISA assays. Sera from 45 patients with Sjogren's syndrome (pSS), 49 with Systemic Lupus Erythematosus (SLE), and 18 with Rheumatoid Arthritis (RA) were tested against the two peptides and the hMBP. Twenty-two per cent of sera from pSS patients, 27% of SLE patients, and none from RA sera reacted with the La epitope; 27% from pSS sera, 22% of SLE sera, and 17% of RA sera gave a positive reaction against the MBP peptide. Finally, 19% of pSS, 30% of SLE, and 38% of RA sera reacted with the hMBP. Thirty-five days after immunization of rabbits with the La epitope, antibodies were produced against all three La/SSB peptides, the MBP peptide, and the hMBP and rechLa proteins. Rabbits immunized with the MBP peptide produced antibodies against the immunizing peptide and the mimicking peptide of La shortly after immunization, whilst antibodies against the other La epitopes and the two intact proteins were produced later. Inhibition experiments in rabbit sera with high reactivity against hMBP, using the MBP peptide as inhibitor, revealed that 80% of serum reactivity was abolished. In conclusion, a significant proportion of human autoimmune sera reacted with both La and MBP derived peptides, as well as with hMBP. La 147-154aa peptide, when used for animal immunizations, induces a fast epitope spreading involving both La and MBP. In contrast, the mimicking MBP epitope induces a delayed response against the other La epitopes. Thus, despite the fact that these peptides present molecular similarity, they induce different immune responses.     

Tula Virus Infection Associated with Fever and Exanthema After a Wild Rodent Bite

Reported here is the first case of human acute infection with Tula virus, which occurred in a 12-year-old boy in Switzerland. This hantavirus had been considered apathogenic to humans, and in Switzerland only TULV-genome sequences have been demonstrated in wild rodents to date. In this case, paronychia, fever and exanthema occurred after the patient was bitten by a wild rodent, indicating an unusual route of hantavirus transmission. Thus, Tula virus infection should be taken into account in patients with appropriate clinical symptoms and contact with rodents. Electronic Publication     

The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses

The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.     

Presence of antisperm antibodies reactive with peptide epitopes of FA-1 and YLP12 in sera of immunoinfertile women.

PROBLEM: Recent studies in several laboratories are focused on delineating sperm antigens that are relevant to fertility and examining involvement of antibodies to these antigens in human immunoinfertility. Our laboratory has characterized two such antigens, namely fertilization antigen (FA-1) and YLP12 dodecamer sequence that are involved in sperm-oocyte binding. The present study was conducted to examine the occurrence of isoantibodies to various peptide epitopes of human and murine FA-1 antigen and YLP12 peptide in sera of immunoinfertile and fertile women. METHOD OF STUDY: Sera from 67 immunoinfertile and 19 fertile women were collected. Various peptides based up on human and murine FA-1 antigen and YLP(12) were synthesized, and examined for immunoreactivity with these sera by using ELISA. Four immunodominant sequences, two each from human (hFA-1 82-97aa and hFA-1 200-219aa) and mouse (mFA-1 2-19aa and mFA-1 117-136aa) FA-1 antigen, were selected for the present study. Another human FA-1 sequence, hFA-1 220-240aa, that was not in the immunodominant region was used as a control. RESULTS: For human FA-1 peptides, 41.8% of the immunoinfertile sera reacted positively (>or=2 SD units) with hFA-1 82-97aa, 24.6% (16/65) with hFA-1 200-219aa, and 3% (2/66) with hFA-1 220-240aa peptide. For two murine FA-1 peptides, 41.7% (25/60) of the immunoinfertile sera reacted positively with mFA-1 2-19aa, and 41.5% (27/65) with mFA-1 117-136aa peptide. For the YLP12 dodecamer peptide, 43.3% (29/67) of the immunoinfertile sera reacted positively. None of the sera from fertile women reacted positively with any of these peptides. CONCLUSION: In conclusion, our data indicate that the immunoinfertile women have circulating isoantibodies against at least two immunodominant peptide epitopes of human and murine FA-1 antigen and YLP12 peptide sequence. These peptides may find clinical application in the specific diagnosis and treatment of female infertility and contraceptive vaccine development.     

Antiviral effect of human saliva against hantavirus

Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome are zoonotic diseases caused by rodent borne hantaviruses. Transmission to humans occurs usually by inhalation of aerozolized virus‐contaminated rodent excreta. Although human‐to‐human transmission of Andes hantavirus has been observed, the mode of transmission is currently not known. Saliva from Puumala hantavirus (PUUV)‐infected patients was shown recently to contain viral RNA. To test if human saliva interferes with hantavirus replication, the effect of saliva and salivary proteins on hantavirus replication was studied. It was observed that saliva from healthy individuals reduced Hantaan hantavirus (HTNV) infectivity, although not completely. Furthermore, HTNV was resistant against the antiviral capacity of histatin 5, lysozyme, lactoferrin, and SLPI, but was inhibited by mucin. Inoculation of bank voles (Myodes glareolus) with HFRS‐patient saliva, positive for PUUV‐RNA, did not induce sero‐conversion. In conclusion, no evidence of infectious virus in patient saliva was found. However, the in vitro experiments showed that HTNV, the prototype hantavirus, is insensitive to several antiviral salivary proteins, and is partly resistant to the antiviral effect of saliva. It therefore remains to be shown if human saliva might contain infectious virions early during infection, that is, before seroconversion. J. Med. Virol. 80:2122–2126, 2008. © 2008 Wiley‐Liss, Inc.     

Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests.     

双抗原夹心法ELISA在检测抗肾综合征出血热总抗体中的应用

目的建立可以检测不同来源血清中抗汉坦病毒抗体的简单、灵敏的方法。方法汉坦病毒核蛋白重组表达纯化后，同时作为捕获作用的固相抗原和检测作用酶标记抗原，建立检测血清中抗汉坦病毒总抗体的双抗原夹心法ELISA法，并与常用的IFA法进行比较分析。结果检测不同血清时特异性为100％，敏感性高于IFA法4～8倍。且不需考虑更换检测试剂，不同来源的血清样本对检测结果没有明显的影响。结论本方法操作简单，成本低，具有较高的敏感性和特异性，适合用于汉坦病毒感染的监测、调查和临床诊断以及宿主动物间病毒感染流行的调查与监测。     

Characterization of cross-reactive and serotype-specific epitopes on the nucleocapsid proteins of hantaviruses

The hantavirus nucleocapsid (N) protein fulfills several key roles in virus replication and assembly and is the major antigen in humoral immune responses in humans and mice. Here we report on epitopes involved in serotype-specific and cross-reactive recognition of the N proteins of hantaviruses using monoclonal antibodies (mAbs) against the N proteins of Andes virus (ANDV) and Sin Nombre virus (SNV). The mAbs define at least twelve different epitopic patterns which span eight sequences, including amino acids 17-59, 66-78, 79-91, 157-169, 222-234, 244-263, 274-286 and 326-338 on the SNV and ANDV N proteins. Studies on the cross-reactivity of these mAbs with different hantavirus N proteins indicated that epitopes located within amino acids 244-286 are related to serotype specificity. We analyzed further the location of epitopes with available three-dimensional structure information including the N-terminal coiled-coil and derived exposed and hidden residues of these epitopes. The generated recombinant N proteins and the characterized mAbs are functional tools being now available for hantavirus diagnostics and replication studies.     

Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents.     

Analysis of the Neutralizing Antibody Response to the Measles Virus Using Synthetic Peptides of the Haemagglutinin Protein

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310–325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35–58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.     

Antigenic structure analysis of glycosylated protein 3 of porcine reproductive and respiratory syndrome virus

The function of the glycosylated protein 3 (GP3), a porcine reproductive and respiratory syndrome virus (PRRSV) associated protein is poorly known. In the present study, the gene encoding GP3 (ORF3), lacking the highly hydrophobic domain in the N- and C-termini was expressed as GST-fusion proteins in E. coli. Monoclonal antibodies (MAbs) against GP3 were developed and used to probe a series of GP3 peptides using ELISA. After precise analysis by sequential deletion of the terminal amino acid residues from each peptide, the minimal epitopes recognized by the MAbs were localized to W(74)CRIGHDRCGED(85) and Y(67)EPGRSLW(74). The epitope sequences were well conserved among most of the North American-type isolates, with the exception of two amino acid mutations in both epitopes in a few of these isolates. Mutational analysis revealed that these mutants were not recognized by any of the five MAbs, indicating that genetic variation could lead to altered antigenicity. Eight out of nine peptide fragments, 58-72aa, 73-87aa, 88-101aa, 102-115aa, 50-65aa, 66-81aa, 80-95aa and 94-109aa were recognized by PRRSV-positive pig serum as determined by Western blot analysis. The results herein may elucidate partially the antigenic structure of GP3 and variations of PRRSV.     

Human recombinant neutralizing antibodies against hantaan virus G2 protein

Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients.