Identification of epitopes on the envelope (E) protein of dengue 2 and dengue 3 viruses using monoclonal antibodies

Summary.  Of a panel of forty-six anti-dengue 3 monoclonal antibodies (MAbs) only three neutralised infection of BHK cells by dengue 3 virus. Attempts to select neutralisation escape mutants (n.e.m.) with two of these antibodies failed. The n.e.m. population selected in the presence of the third neutralising antibody, 1H9, had a nucleotide change at position 1157 of the E protein gene resulting in a non-conservative amino acid change at E386 for a Lys to an Asn. A dengue 2 n.e.m. was selected with the flavivirus crossreactive IgG monoclonal antibody 4G2, had deduced amino acid changes at E169 (Ser to Pro) and E275 (Gly to Arg). This dengue 2 n.e.m. population produced smaller plaques in BHK cells than the parental virus, decreased fusion activity (FFWI) and had lost the ability to agglutinate gander erythrocyes at pH 6.0 to 6.7. Received February 28, 2001 Accepted July 6, 2001     

Analysis of functional epitopes on the dengue 2 envelope (E) protein using monoclonal IgM antibodies

Summary Forty-two hybridomas secreting IgM antibody against dengue virus were derived from spleen cells of dengue 2 infected mice. Antibody from 27 of these recognised the E protein of this virus. Of the 22 antibodies which neutralised dengue 2, only two cross-reacted with other flaviviruses. These 22 antibodies recognised three discrete domains on dengue virions. Competitive binding studies with IgG monoclonal antibodies suggested that two of the three domains were recognised by both IgG and IgM antibodies and that there were two additional domains which may be recognised exclusively by either IgG or IgM antibodies. IgM antibodies reacting with domains recognised by IgG antibodies that enhanced infection of susceptible cells by dengue 2, had no enhancing properties. None of the IgM antibodies activated the serum complement system after reacting with dengue 2 virions.     

Epitopes on the dengue 1 virus envelope protein recognized by neutralizing IgM monoclonal antibodies

Three of 41 IgM monoclonal antibodies derived from dengue 1 virus immunized mice neutralized dengue 1 infection in vitro. All three neutralizing monoclonal antibodies reacted with spatially related epitopes on the E protein of dengue 1 which were also recognized by antibodies in sera from dengue patients. Two neutralization-resistant populations of dengue 1 virus, D1-M10 and D1-M17, were selected by sequential passage of virus in C6/36 cells in the presence of neutralizing IgM monoclonal antibodies M10 and M17, respectively. Single nucleotide changes occurred in the E protein gene of each of these virus populations resulting in single amino acid substitutions at E279 (Phe-Ser) in D1-M10 and at E293 (Thr-Ile) in D1-M17. Both neutralization-resistant populations of virus were more sensitive to elevated temperature than was the wild-type dengue 1 virus and the infectivity and haemagglutinating ability of the neutralization-resistant populations decreased more slowly than that of wild-type virus when exposed to pH in the range 5.8 to 7.0. These are the first epitopes involved in neutralization to have been identified in dengue 1 virus and the first outside domain III of the E protein on any dengue virus.     

Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies

Summary To investigate the role of Hantaan virus envelope glycoprotein in infection, a panel of monoclonal antibodies (MAbs) was examined in vitro with several serological tests and in vivo by passive transfer experiments in mice. An antigenic site, specific for the inhibition of infected cell focus was detected with the focus inhibition neutralization test (FINT), in addition to the neutralization related antigenic sites, which were revealed by the ordinary focus reduction neutralization test (FRNT). Suckling mice were given the MAbs by passive transfer followed by lethal Hantaan virus challenge. All neutralizing MAbs detected by either FRNT or FINT protected all mice from lethal infection, confirming the importance of the antigenic sites as a protective antigen. Mice given non-neutralizing MAbs by passive transfer, however, began to die earlier than the control group; mean time to death (18.2±2.1 to 21.5±2.8 days) being significantly shorter than that of the control group (25.8±1.8, p<0.01, Mann-Whitney,U probability test). Virus titers in brains of mice which died early, were about 10 times higher than those of control mice. These results indicated the early death phenomenon of mice which was mediated by the antivirus antibody.     

Epitopes on the peplomer protein of infectious bronchitis virus strain M41 as defined by monoclonal antibodies

Sixteen monoclonal antibodies (Mcabs) were prepared against infectious bronchitis virus strain M41, all of them reacting with the peplomer protein. One of them, Mcab 13, was able to neutralize the virus and to inhibit hemagglutination. Competition binding assays allowed the definition of five epitopes, designated as A, B, C, D, and E, of which epitopes A and B are overlapping. Furthermore, the binding of Mcab 13 (epitope E) could be enhanced by the addition of Mcabs from group B, C, and D. A dot immunoblot assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. Only the binding of Mcab 13 was affected, indicating that the epitope involved in neutralization and hemagglutination is conformation dependent. The epitopes A to D were highly conserved among IBV strains, while epitope E was specific for strains M41 and D3896. In this last strain, however, this epitope was not involved in neutralization.     

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.     

Single antigen detects both immunoglobulin M (IgM) and IgG antibodies elicited by all four dengue virus serotypes

The resurgence of dengue (DEN) virus infections in the last few decades coupled with the lack of a preventive vaccine and specific antiviral drugs has jointly contributed to making this a significant global public health problem. Currently, symptomatic supportive treatment and fluid replacement therapy are the only means available to minimize DEN-induced mortality. As the clinical symptoms associated with DEN virus infections are indistinguishable from those of many other viral, bacterial, and parasitic infections, specific diagnostic tests assume critical importance in the unequivocal identification of DEN virus infections. We have designed a novel chimeric antigen based on envelope domain III (EDIII), a critical antigenic region of the major structural protein of DEN viruses. We fused EDIIIs corresponding to each of the four DEN virus serotypes using pentaglycyl linkers, overexpressed the resultant tetravalent chimeric protein in Escherichia coli, and affinity purified it in high yields, obtaining approximately 30 mg protein of >95% purity per liter of culture. We show that this tetravalent antigen could specifically recognize anti-DEN virus antibodies of both the immunoglobulin M (IgM) and IgG classes. Using a large panel of IgM antibody capture-enzyme-linked immunosorbent assay- and hemagglutination inhibition-confirmed DEN virus-infected and uninfected patient sera (n = 289), we demonstrate that this tetravalent antigen can function as a diagnostic tool of high sensitivity and specificity.     

Identification of an immunodominant region on the isolated bovine leukaemia virus (BLV) major envelope protein gp51 by monoclonal antibodies presumably not exposed during natural BLV infection

A panel of newly isolated monoclonal antibodies (MoAbs) is described which are specific for bovine leukaemia virus (BLV) envelope protein gp51. Epitope mapping using competition antibody binding assays and binding studies with gp51-related fusion proteins and synthetic peptides show that they are directed against seven independent antigenic determinants. Four of them are unrelated to the epitopes described earlier (Bruck et al., 1982a). We define three binding regions for the MoAbs on the gp51 molecule. The region between amino acids 170 and 217 is highly immunogenic when the isolated protein is used for immunization, and seems to be inaccessible for immune recognition when gp51 is associated with the virus envelope as it occurs during natural BLV infection.     

Isolation and characterization of monoclonal antibodies specific for protein conformational epitopes present in prostate-specific membrane antigen (PSMA)

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.     

Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins.

Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.     

Identification of a linear epitope on the haemagglutinin protein of pandemic A/H1N1 2009 influenza virus using monoclonal antibodies

A novel influenza A/H1N1 virus, emerging from Mexico and the United States in the spring of 2009, caused the pandemic human infection of 2009-2010. The haemagglutinin (HA) glycoprotein is the major surface antigen of influenza A virus and plays an important role in viral infection. In this study, three hybridoma cell lines secreting specific monoclonal antibodies (Mabs) against the HA protein of pandemic influenza A/H1N1 2009 virus were generated with the recombinant plasmid pCAGGS-HA as an immunogen. Using Pepscan analysis, the binding sites of these Mabs were identified in a linear region of the HA protein. Further, refined mapping was conducted using truncated peptides expressed as GST-fusion proteins in E. coli. We found that the ²⁵⁰VPRYA²⁵⁴ motif was the minimal determinant of the linear epitope that could be recognized by the Mabs. Alignment with sequences from the databases showed that the amino acid residues of this epitope were highly conserved among all pandemic A/H1N1 2009 viruses as well as the classical swine H1N1 viruses isolated to date. These results provide additional insights into the antigenic structure of the HA protein and virus-antibody interactions at the amino acid level, which may assist in the development of specific diagnostic methods for influenza viruses.     

Definition of an epitope on Japanese encephalitis virus (JEV) envelope protein recognized by JEV-specific murine CD8+ cytotoxic T lymphocytes

Summary.  We defined an epitope on the Japanese encephalitis virus (JEV) envelope (E) protein recognized by CD8⁺ cytotoxic T lymphocytes (CTLs). CTLs induced in JEV-infected BALB/c (H-2^d) mice recognized E and/or premembrane (PrM) proteins, while CTLs in C57BL/6J (H-2^b) and C3H/HeJ (H-2^k) mice did not. JEV-specific CTLs had a phenotype of CD3⁺ CD4⁻ CD8⁺. Twenty-four 9-amino acid (a.a.) peptides, which had binding motifs for H-2K^d, H-2L^d or H-2D^d, were synthesized according to the amino acid sequences of PrM and E proteins. CTLs induced by JEV infection recognized only the peptide K-3. Immunization of BALB/c mice with only a group of peptides including K-3 induced CTLs which recognized the homologous K-3 peptide, while immunization with other peptides did not. The peptide K-3 had a binding motif for H-2K^d. This is consistent with the finding that JEV-specific CTLs in BALB/c mice was H-2K^d-restricted. These results indicate that the epitope recognized by CTLs in BALB/c mice is located between a.a. 60 and 68 on the E protein, corresponding to an a.a. sequence of CYHASVTDI. Accepted September 29, 1999     

Identification of a conserved linear epitope on the VP1 protein of serotype O foot-and-mouth disease virus by neutralising monoclonal antibody 8E8

Foot-and-mouth disease virus (FMDV) serotype O remains an important threat to animal husbandry worldwide, and the variability of the virus presents a major problem for FMDV vaccine design. High-affinity neutralising antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype O and protect against future pandemics. We generated a novel monoclonal antibody (MAb) 8E8 that potently neutralised infection of FMDV O/YS/CHA/05 both in vitro and in vivo. Screening of a phage-displayed random 12-peptide library revealed that MAb 8E8 bound to phages displaying a consensus motif GDLNVRT, which is highly homologous to (146)GDLQVLT(152) of the FMDV VP1 protein. Given that MAb 8E8 showed reactivity with the (146)GDLQVLT(152) motif, we proposed that this motif represented a linear B-cell epitope of the VP1 protein. Western blot analysis revealed that the epitope peptide could be recognised by the positive sera from serotype O FMDV-infected pigs. The (147)DLQVLT(152) motif was the minimal requirement for reactivity as demonstrated by reactivity of MAb 8E8 with several truncated peptides derived from the motif. For further mapping, a set of different extended motifs derived from the minimally reactive epitope was expressed with a GST-tag and subjected to western blot. The results showed that a 10-aa peptide (145)RGDLQVLTPK(154) was the minimal unit with maximal binding activity to MAb 8E8. Subsequent alanine scanning mutagenesis studies revealed that D(147), Q(149) and V(150) are crucial for MAb 8E8 binding. Furthermore, the epitope was found to be highly conserved among different topotypes of serotype O FMDV through sequence alignment analysis and detection of MAb 8E8 for affinity to some isolates collected in China. Thus, the 8E8 epitope identified here should be helpful for designing epitope-based, intra-typic, cross-protective vaccines of serotype O FMDV.     

Detection of hepatitis G virus (HGV) RNA and antibodies to the HGV envelope protein E2 in a cohort of hemodialysis patients

An analysis of the evolution of hepatitis G virus (HGV) infection markers was performed for a cohort of 58 hemodialyzed patients. During follow-up (4.88 +/- 0.42 years), a group of these patients cleared their antibodies against the envelope protein E2 with (4 of 29 cases; 13.8%) or without (9 of 29 cases; 31%) the reappearance of viremia. This finding implies a temporally limited protection in patients previously infected with HGV.     

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ( approximately 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.     

IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein

To develop an enzyme immunoassay (EIA) for IgM antibody to hepatitis E virus (HEV) (IgM anti-HEV) and IgG antibody to HEV (IgG anti-HEV), a synthetic gene encoding several liner immuno-dominant antigenic epitopes from HEV structural proteins was assembled as a chimeric recombinant mosaic protein (Mpr) with glutathione S-transferase and used as an immunodiagnostic target. In addition, a neutralization confirmation test was developed using individual synthetic peptides. Among 614 patients with acute hepatitis from 10 geographically distinct outbreaks, IgG anti-HEV was found in 546 (88.9%), with a range of 77–100% depending on the outbreak. Of 130 patients tested for IgM anti-HEV, 126 (96.9%) were positive. Among patients tested within 4 months of onset of jaundice, 37/37 (100%) were IgG anti-HEV positive. For patients from whom sera were collected 1–16 days after onset of jaundice, the geometric mean IgG titer (GMT) was 1:47,000; the GMT increased to 1:70,710 30–40 days after onset of jaundice and decreased to 1:1,778 3–4 months after the onset of jaundice. For patients tested 6–8 months after onset of jaundice, 11/12 (92%) were IgG anti-HEV positive, and the GMT was 1:2,908. IgM anti-HEV was detected in 43/43 (100%) sera collected 1–40 days after onset of jaundice, and the GMT for IgM anti-HEV was 1:10,000 at that time. For sera collected 3–4 and 6–12 months after onset of jaundice, 7/14 (50%) and 5/12 (40%) respectively, were IgM anti-HEV positive. In conclusion, an artificial mosaic protein composed of linear antigenic epitopes from open reading frame 2 (ORF2) and ORF3 of HEV has been successfully applied to the development of a sensitive and specific EIA for the detection of IgG and IgM anti-HEV activity. These assays were used for the verification of HEV infection in outbreak settings and for the diagnosis of HEV infection in sporadic cases. © 1996 Wiley-Liss, Inc.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.