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.

Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.

Idiotypic-anti-idiotypic B cell interactions generated against a protective antigen of a morbillivirus in mice.

The idiotypic network theory (N. K. Jerne, Ann. Immunol. 125, 373-389, 1974) predicts that any antibody that can be made by an individual would have its preexisting specific complementary B cells in its germline repertoire. We transplanted syngeneic BALB/c mice with live hybridoma cells and demonstrated the simultaneous presence of interacting idiotypic and anti-idiotypic B cells in an individual animal by immuno-cytoadherence assays. Furthermore, we demonstrate that interacting B cells displaying idiotypic and anti-idiotypic antibodies are subjected to lysis by complement. It is therefore tempting to speculate that this complement-sensitive interaction between idiotypic and complementary anti-idiotypic B cells in vivo may provide a mechanism for the regulation of B cell populations.

Perpetuation of immunological memory: a relay hypothesis.

A mechanism is proposed which explains the perpetuation of B-cell immunological memory indefinitely without requiring the presence of long-living memory cells or persisting antigen. The salient feature of this model is that immunological memory can be perpetuated indefinitely through the mutual interaction of idiotypic and anti-idiotypic B cells. These cells mutually stimulate and clonally expand with either specific or bystander T-cell help. Because B cells can present antigen, they present 'apparently foreign' idiopeptides to T cells. The idiopeptides of de novo synthesized antibody is presented to CD8+ T cells that recognize the idiopeptide-presenting cell as targets and regulate their population. The recycling of immunoglobulins from surface to endosomal compartment of B cells leads to the presentation of idiopeptides by major histocompatibility complex (MHC) class II to CD4+ T cells. Even if the majority of the clonally expanded cells die because of lack of stimulation, cytotoxic T lymphocyte (CTL) lysis or for other reasons, the surviving cells will be able to carry forward the memory. This mechanism also provides a means for affinity maturation through idiotypic selection of somatically mutated high affinity cells or those from the naïve pool. We have termed these two types of complementary B cells as Burnet B cells: those which recognize the antigen or antigen mimic, and Jerne B cells, which can recognize the idiotypes of antibody and carry antigen mimics. The proposed hypothesis can explain differential duration of memory for different antigens, the shelf space paradox, affinity maturation, repertoire shift, etc.

Identification of a cytotoxic T-cell epitope on the recombinant nucleocapsid proteins of Rinderpest and Peste des petits ruminants viruses presented as assembled nucleocapsids.

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.