Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): identification of neutralizing and antibodies reactive to S, N, M and E viral proteins

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。     

Development and application of monoclonal antibodies against avian influenza virus nucleoprotein

Rapid and accurate diagnosis of avian influenza (AI) infection is important for an understanding epidemiology. In order to develop rapid tests for AI antigen and antibody detection, two monoclonal antibodies (mAbs) against influenza nucleoprotein (NP) were produced. These mAbs are designated as F26-9 and F28-73 and able to recognize whole AI virus particles as well as the recombinant NP. Both of the mAbs were tested in a slot blot for their reactivity against 15 subtypes of influenza virus; F28-73 reacted with all tested 15 subtypes, while F26-9 failed to react with H13N6 and H15N8. The mAb binding epitopes were identified using truncated NP recombinant proteins and peptide array techniques. The mAb F26-9 reacted with NP-full, NP-1 (638bp), NP-2 (315bp), NP-4 (488bp), and NP-5 (400bp) in the Western blot. The peptide array results demonstrated that the mAb F26-9 reacted with NP peptides 15-17 corresponding to amino acids 71-96. The mAb F28-73 recognized the NP-full, -1 and -4 fragments, but failed bind to NP-2, -3, -5, and any peptides. This antibody-binding site is expected to be contained within 1-162 amino acids of AI NP, although the exact binding epitope could not be determined. The two mAbs showed reactivity with AI antigen in immunofluorescence, immunohistochemistry and immune plaque assays. Immune response of AI infected animals was determined using the mAb F28-73 in a cELISA. All tested chickens were positive at 11 days post-infection and remained positive until the end of the experiment on day 28 (>50% inhibition). The two mAbs with different specificities are appropriate for developing various tests for diagnosis of AI infection.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Detection of antibodies against human metapneumovirus by Western blot using recombinant nucleocapsid and matrix proteins

Detection of antibodies against individual proteins of human metapneumovirus (hMPV) is important in the analysis of immune responses to hMPV. Specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. The results were compared with those of immunofluorescence assays (IFAs) based on hMPV-infected LLC-MK2 cells, which expressed the whole hMPV proteins. Thirty (61.2%) and 31 (63.3%) of 49 serum samples with titers of > or = 1:160 by IFA reacted with N and M proteins, respectively. Only 2 (4.2%) of 11 serum samples with titers of 1:80 by IFA reacted with N and M proteins. Antibodies against N and M proteins were not detected in 37 serum samples with titers of < 1:40 by IFA. These results indicate that the antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1%, N protein; 40.8%, M protein) than those against whole proteins of hMPV detected by IFA. The reactivity of sera with the recombinant N protein and that with the recombinant M protein correlated well (correlation coefficient of 0.79), and the concordance of reactivities was 91% (kappa = 0.79). In summary, both recombinant N and M proteins of hMPV were antigenic, and the responses to N and M protein varied among patients. Therefore, Western blot using N and M proteins provide a useful tool for analysis of immune responses to hMPV.     

Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus

We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.     

Characterization of monoclonal antibodies against hantavirus nucleocapsid protein and their use for immunohistochemistry on rodent and human samples

Monoclonal antibodies are important tools for various applications in hantavirus diagnostics. Recently, we generated Puumala virus (PUUV)-reactive monoclonal antibodies (mAbs) by immunisation of mice with chimeric polyomavirus-derived virus-like particles (VLPs) harbouring the 120-amino-acid-long amino-terminal region of the PUUV nucleocapsid (N) protein. Here, we describe the generation of two mAbs by co-immunisation of mice with hexahistidine-tagged full-length N proteins of Sin Nombre virus (SNV) and Andes virus (ANDV), their characterization by different immunoassays and comparison with the previously generated mAbs raised against a segment of PUUV N protein inserted into VLPs. All of the mAbs reacted strongly in ELISA and western blot tests with the antigens used for immunization and cross-reacted to varying extents with N proteins of other hantaviruses. All mAbs raised against a segment of the PUUV N protein presented on chimeric VLPs and both mAbs raised against the full-length AND/SNV N protein reacted with Vero cells infected with different hantaviruses. The reactivity of mAbs with native viral nucleocapsids was also confirmed by their reactivity in immunohistochemistry assays with kidney tissue specimens from experimentally SNV-infected rodents and human heart tissue specimens from hantavirus cardiopulmonary syndrome patients. Therefore, the described mAbs represent useful tools for the immunodetection of hantavirus infection.     

Development,characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

Ebola virus (EBOV) causes uncommon but dramatic outbreaks in remote regions of Africa, where diagnostic facilities are limited. In order to develop diagnostic tests, which can be handled and distributed easily, monoclonal antibodies (mAbs) to EBOV, species Zaire, were produced from mice immunized with inactivated viral particles. Nine stable hybridoma cell lines were obtained producing specific mAbs directed against the viral structural protein VP40. These mAbs were characterized by enzyme-linked immunosorbent, immunoblot and immunofluorescence assays. Subsequently, an antigen capture enzyme-linked immunosorbent assay was established, which detects VP40 of all known species of EBOV. This assay could detect viral material in spiked human serum that has been sodium dodecylsulfate-inactivated. The established enzyme-linked immunosorbent assay therefore has the ability to become a very useful tool for obtaining an accurate diagnosis in the field, limiting the risk of laboratory infections.     

Generation and characterisation of monoclonal antibodies against influenza virus A, subtype H5N1

The emergence of highly pathogenic avian influenza A virus (HPAIV) subtype H5N1 in 1997 has since resulted in large outbreaks in poultry and in transmission from poultry to humans, mostly in southeast Asia, but also in several European countries. Effective diagnosis and control measures are essential for the management of HPAIV infections. To develop a rapid diagnostic test, a panel of murine monoclonal antibodies (mAbs) against influenza virus A subtype H5 was generated. Eleven mAbs were produced and characterised according to their reactivity by indirect and sandwich ELISA and western blotting against different H5 subtypes representing past and viruses currently circulating. Ten out of 11 mAbs reacted strongly with the haemagglutinin (HA) protein of H5 viruses, whereas one mAb reacted with the M1 protein. Targeted HA protein epitopes seemed to be conformational. One hybridoma clone binds to a linear epitope of the M1 protein. One specific mAb reacts with HPAIV H5 in the immunofluorescence test, and two antibodies neutralised H5 viruses. On the basis of the results, the set of seven mAbs is appropriate for developing diagnostic tests. With the generated mAbs, a sandwich ELISA was developed recognising all H5N1 strains tested but no other influenza viruses. With this ELISA, as little as 0.005 HA units or 0.1 ng/ml H5N1 was detected, surpassing other ELISA tests. The novel reagents have the potential to improve significantly available rapid antigen detection systems.     

Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed.

The peplomer protein (S) and the transmembrane protein (M) of transmissible gastroenteritis virus (TGEV) of swine were identified by iodination and serologically on the surface of infected cells. Of a total of 4 monoclonal antibodies (mAb) directed against four antigenic sites of S protein (Correa et al., 1988), 3 specific for sites A, B and D attached to the plasma membrane of infected cells, as disclosed by indirect immunofluorescence and by complement-mediated cytolysis. Four of the mAbs assayed were specific for the viral protein M and two of them gave plasma membrane immunofluorescence and mediated cytolysis in the presence of complement. The viral nucleoprotein N could not be demonstrated on the surface of infected cells either by iodination or employing 3 mAbs against this protein. Finally, a time course infection experiment demonstrated that S and M proteins were expressed on the surface of infected cells at 4 h after infection, before infective virus was released from infected cells.     

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.     

Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China

Lymphocystis disease virus (LCDV) can infect, both naturally and experimentally, about 100 different teleost fish species. In this study, LCDV was purified using differential and gradient centrifugation from skin tumours of Japanese flounder Paralichthys olivaceus. A panel of five monoclonal antibodies (Mabs) against LCDV were produced by immunization of Balb/c mice with purified virus preparations. Analysed by the indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), Western blot and immunogold electron microscopy (IEM), they showed specificity for LCDV. Immunofluorescent studies showed that the specific fluorescence signals appeared at the peripheral zone of hypertrophied cells cytoplasm where was the cytoplasmic inclusion bodies location and many of them formed ribbon-shaped. Western blot analysis demonstrated that two Mabs 1D7 and 2B6 reacted specifically to a single protein with an approximately molecular weight of 116kDa, Mab 3G3 reacted with two LCDV proteins at molecular mass of approximately 116 and 90kDa. Immunogold transmission electron-microscopy provided visualized evidence that the epitopes recognized by these Mabs were located on the outer surface of virions. The Mabs characterized should prove useful for developing LCDV diagnostic assays and for studying the biology of infection and pathogenesis of disease.     

Production and diagnostic application of monoclonal antibodies against influenza virus H5

Nine monoclonal antibodies (mAbs) against avian influenza virus (AI) H5 subtype from mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/66) were produced. Upon testing, the results indicated that the binding epitopes of eight out of the nine mAbs were conformational, while one mAb (#7) reacted with denatured H5N1 only. Two mAbs #10 and #11 reacted with all of the thirteen H5 strains tested indicating that the binding epitopes of these mAbs were conserved among these H5 subtypes.Possible applications of these mAbs in rapid tests for H5 antigen were explored. Double antibody sandwich (DAS) ELISAs were developed using two selected mAbs #10 and #11. This DAS ELISA detects specific H5 viruses and is able to identify all thirteen H5 strains tested. Three mAbs showed reactivity with AI H5 antigen for both immunofluorescence (IF) and immunohistochemistry. A cELISA used to screen chickens that had been infected with an H5 virus was developed with mAb #9 and recombinant H5 antigen. The sera from chickens that have been infected with an H5N1 virus were examined using the cELISA. 80% of the sera from H5 infected chickens showed a positive H5 specific antibody response at 7 days post-infection (dpi) and remained positive until the end of the experiment on day 30 (>40% inhibition). This panel of the AI H5 specific mAbs is valuable for the development of various immunoassays.     

Monoclonal antibodies against the nucleocapsid proteins of henipaviruses: production, epitope mapping and application in immunohistochemistry

Summary Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library revealed two linear antigenic sites of the henipavirus N proteins, KLxR (aa 17–20) and FKREM (aa 446–450), which have not been reported previously. Two of the mAbs were able to specifically recognize HeV antigens by immunohistochemical staining of lung tissue sections of a horse experimentally infected with HeV. These reagents will be a useful addition to the collection of tools essential for further research and improvement in diagnosis of henipaviruses. Correspondence: Changchun Tu, Institute of Veterinary Sciences, Academy of Military Medical Sciences, 1068 Qinglong Road, Changchun 130062, China     

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) Rhabdovirus

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备及初步鉴定

目的 :制备抗禽流感病毒 (AIV)H9亚型血凝素蛋白的单克隆抗体 (mAb)。方法 :以AIVH9亚型油乳剂灭活疫苗作为免疫原 ,免疫 8wk龄BALB/c小鼠。采用淋巴细胞杂交瘤技术制备抗AIVH9亚型血凝素蛋白的mAb ;采用ELISA和血凝抑制试验(HI)检测腹水mAb的效价 ;采用ELISA、HI、免疫荧光染色 (IF)及Westernblot鉴定mAb的特异性。结果 :获得 3株可稳定分泌特异性mAb的杂交瘤细胞株 2A3、2H1和 1C8,其腹水mAb的ELISA效价依次为 1× 10 7、1× 10 5和 5× 10 6,血凝抑制效价为 1× 2 8～ 1× 2 13 ;3株mAb的Ig亚类均为IgG1。以mAb 2H1进行Westernblot的结果显示 ,该mAb能与AIV的Mr 为 75 0 0 0的蛋白条带起反应 ,表明其是针对AIVH9亚型血凝素蛋白的mAb。与 32株AIVH9亚型国内分离株进行血凝抑制试验表明 ,mAb 2H1具有良好的广谱性。结论 :成功地制备了抗AIVH9亚型血凝素蛋白的mAb ,为AIV的抗原性分析、血清学诊断、疫苗质量的监测及流行病学调查等奠定了基础     

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I-III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.     

Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins

Summary.  The protective capacity of monoclonal antibodies (MAbs) generated to the dengue-2 virus envelope (E) and premembrane (prM) proteins was tested in vivo. Two anti-E MAbs, 2C5.1 and 4G2 and two anti-prM MAbs, 2A4.1 and 2H2 provided cross-protection against all four dengue virus serotypes. Overlapping sets of synthetic peptides spanning amino-acid sequence 301–401 (domain III) of the E protein and the entire prM protein were then used to locate their epitopes. The anti-E MAbs strongly reacted with the peptide sequence 349-GRLITVNPIVT-359 (E349–359) from domain III and the immunodominant epitope, 274-SGNLLFTGHL-283 (E274–283) from the hinge region between domains I and II. The anti-prM MAbs strongly reacted with the sequence, 40-PGFTVMAAIL-49 (M40–49) from the first membrane-spanning domain of the M protein. These anti-prM MAbs also reacted with peptides E274–283 and E349–359, while the anti-E MAbs reacted with a peptide sequence, 1-FHLTTRNGEP-10 from the prM protein and these cross-reactions with both proteins were confirmed using immunoblot assays. MAbs 2C5.1, 4G2 and 2H2 more strongly reacted with an MEH1 peptide GLFTPNLITI, which was designed as an antigenic hybrid between these E and prM peptide sequences, than with any of these natural peptide sequences. These peptide sequence will now be tested for their ability to generate cross-protective antibodies against each dengue virus serotype when delivered with appropriate T-helper epitopes. Accepted August 13, 1999