A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.
A standardized serum dilution plaque reduction neutralization test was used for cross-neutralization studies of 23 strains of Bunyamwera serogroup viruses. Antigenic relationships were determined by inspection of the neutralization tests results as well as by numerical taxonomic analysis. Based on these analyses five complexes, containing 1-11 viruses, were distinguished. Little or no cross-reactivity was observed between viruses of different complexes. Three of the viruses tested were indistinguishable from prototypes and probably represent strains or varieties of those prototypes. A tentative classification scheme for the Bunyamwera group is presented.
Background Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE. Methods We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants. Findings Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope. Conclusions The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.
ABSTRACT An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10 2.1 to 10 3.7 PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools ( n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10 3 PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.
A humanized monoclonal antibody (mAb) has been developed and its potential to protect from or cure a Venezuelan equine encephalomyelitis virus (VEEV) infection was evaluated. The VEEV-neutralizing, protective murine mAb 3B4C-4 was humanized using combinatorial antibody libraries and phage-display technology. Humanized VEEV-binding Fabs were evaluated for virus-neutralizing capacity, then selected Fabs were converted to whole immunoglobulin (Ig) G1, and stable cell lines were generated. The humanized mAb Hy4-26C, designated Hy4 IgG, had virus-neutralizing capacity similar to that of 3B4C-4. Passive antibody protection studies with purified Hy4 IgG were performed in adult Swiss Webster mice. As little as 100 ng Hy4 IgG protected 90 % of mice challenged with 100 intraperitoneal (i.p.) mean morbidity (MD(50)) doses of virulent VEEV (Trinidad donkey) 24 h after antibody transfer; also, 500 mug Hy4 IgG protected 80 % of mice inoculated with 100 intranasal MD(50) doses of VEEV. Moreover, 10 mug passive Hy4 IgG protected 70 % of mice from a VEEV challenge dose as great as 10(7) i.p. MD(50). Hy4 IgG also protected mice from challenge with another epizootic VEEV variety, 1C (P676). Importantly, therapeutic administration of the humanized mAb to mice already infected with VEEV cured 90 % of mice treated with Hy4 IgG within 1 h of VEEV inoculation and 75 % of mice treated 24 h after virus infection.
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