T-helper cell epitopes on the E-glycoprotein of dengue 2 Jamaica virus.

To identify T-helper (Th)-cell epitopes, we analyzed 25 synthetic peptides, which included most of the 495-amino-acid sequence of the envelope (E)-glycoprotein of dengue 2 virus. The peptides were analyzed in three mouse strains, BALB/c (H-2d), C57BL/6 (H-2b), and outbred NIH-Swiss, for their ability to elicit antibody or prime the Th-cell compartment following two inoculations in Freund's incomplete adjuvant. Sixteen peptides were able to elicit an antipeptide antibody response in one or more mouse strain. Eleven antipeptide serum pools were able to bind to virus in ELISA. Fifteen peptides primed one or more haplotype for an in vitro antipeptide Th-cell response as measured by blastogenesis. Th-cell activation was generally confirmed by measurable in vitro production of interleukin (IL)-2/IL-4. Nine peptides that were positive for in vitro blastogenesis, 1-2, 35, 4-6, 79, 142, 208, 06, 16, and 17, elicited virus-reactive Th-cells in vitro in H-2d mice. Two of these peptides (4-6 and 17) were able to prime virus-reactive Th-cells in H-2b mice. Nine peptides primed outbred mice in vitro for an antiviral antibody response significantly greater than that seen in animals primed with an irrelevant peptide. These results correlate with, and expand on, our previous observations based on a smaller set of synthetic peptides derived from the E-glycoprotein of Murray Valley encephalitis virus and suggest that synthetic peptides can function as E-glycoprotein Th-cell epitopes. The similarity of results between two distantly related flaviviruses suggests that E-glycoprotein Th-cell epitopes are consistent in location and activity.

A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

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.

Variants of Venezuelan equine encephalitis virus that resist neutralization define a domain of the E2 glycoprotein.

Stable neutralization (N) escape variants of Venezuelan equine encephalitis (VEE) virus were selected by anti-E2 glycoprotein monoclonal antibodies (MAbs) that neutralize viral infectivity, block viral hemagglutination, and passively protect mice. The nucleotide sequence of the E1, E2, and E3 genes of four variants revealed a clustering of single mutations in a domain spanning E2-182 to E2-207. The conformation of this short linear sequence affects antigenicity in the N domain because reduction and alkylation of virus disrupted binding of some E2 neutralizing MAbs. Serologic evidence for interaction of E2 epitopes also was obtained. Mutations in the N domain of VEE virus did not alter the kinetics of binding to Vero cells. They did, in some cases, produce attenuation of virulence in mice.