The value of predictive algorithms for identifying CD8+ T (T(CD8+))-cell epitopes has not been adequately tested experimentally. Here we demonstrate that conventional bioinformatic methods predict the vast majority of T(CD8+)-cell epitopes derived from vaccinia virus WR strain (VACV-WR) in the H-2(b) mouse model. This approach reveals the breadth of T-cell responses to vaccinia, a widely studied murine viral infection model, and may provide a tool for developing comprehensive antigenic maps of any complex pathogen.

Innate immune responses to vaccine adjuvants based on lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, are driven by Toll-like receptor (TLR) 4 and adaptor proteins including MyD88 and TRIF, leading to the production of inflammatory cytokines, type I interferons, and chemokines. We report here on the characterization of a synthetic hexaacylated lipid A derivative, denoted as glucopyranosyl lipid adjuvant (GLA). We assessed the effects of GLA on murine and human dendritic cells (DC) by combining microarray, mRNA and protein multiplex assays and flow cytometry analyses. We demonstrate that GLA has multifunctional immunomodulatory activity similar to naturally-derived monophosphory lipid A (MPL) on murine DC, including the production of inflammatory cytokines, chemokines, DC maturation and antigen-presenting functions. In contrast, hexaacylated GLA was overall more potent on a molar basis than heterogeneous MPL when tested on human DC and peripheral blood mononuclear cells (PBMC). When administered in vivo, GLA enhanced the immunogenicity of co-administered recombinant antigens, producing strong cell-mediated immunity and a qualitative TH1 response. We conclude that the GLA adjuvant stimulates and directs innate and adaptive immune responses by inducing DC maturation and the concomitant release of pro-inflammatory cytokines and chemokines associated with immune cell trafficking, activities which have important implications for the development of future vaccine adjuvants.

Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.

Vaccinia virus (VACV) affords long-lasting protection against variola virus, the agent of smallpox. VACV-reactive CD8 T cells contribute to protection but their molecular control is unknown. We show that the TNFR molecule OX40 (CD134) controls primary VACV-specific CD8 T cell expansion and antiviral cytokine production and dictates development of strong memory to both dominant and subdominant VACV epitopes. Using adoptive transfer of OX40-deficient CD8 TCR-transgenic T cells responding to Ag in the context of VACV infection, we found that this reflects a direct action of OX40 expressed by CD8 T cells. Furthermore, CD8 T cells that can protect against lethal VACV challenge do not develop in mice deficient in OX40. Thus, OX40, which has been found to play little if any role in the generation of CD8 T cells to several viruses, including lymphocytic choriomeningitis virus and influenza, plays a dominant role in shaping the CD8 T cell response to VACV. These data suggest that unique costimulatory pathways might control alternate antiviral CD8 responses, demonstrating the plasticity of the immune response in utilizing different mechanisms to achieve similar ultimate goals.

Recent studies have defined vaccinia virus (VACV)-specific CD8؉ T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4 ؉ T cell responses. In this study, we identified 14 I-A b -restricted VACV-specific CD4 ؉ T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for ϳ20% of the total anti-VACV CD4 T he CD4ϩ T cells play a central role in the host defense against invading pathogens. Several recent studies showed a critical role of CD4 ϩ T cells for eliciting an efficient memory CD8 ϩ T cell response against acute viral infections. Memory CD8 ϩ T cells primed in the absence of CD4 ϩ T cell help are qualitatively impaired in their ability to mount a vigorous response to secondary encounter with Ag (1-4). CD4ϩ T cells also help B cells to produce neutralizing Abs and play a critical role in generating B cell memory (5, 6). Finally, CD4ϩ T cells can directly impede the spread of viruses through the secretion of antiviral cytokines such as IFN-␥ that block viral replication, and cytotoxic functions have also been attributed to CD4 ϩ T cells (7,8).A clear role of CD4 ϩ T cells is apparent in infection with vaccinia virus (VACV) 3 . Immunization with VACV induces cellular immune responses that persist for Ͼ35 years (5, 9 -11). T cell immunity is most important for VACV resistance in naive mice (12). In contrast, Ab responses play a major role in protection against lethal challenge with VACV in immunized mice (12) or monkeypox virus in primates (13).In a recent study, a single modified vaccinia virus Ankara (MVA) inoculation protected mice deficient of B and CD8 ϩ T cells against lethal VACV intranasal challenge, whereas CD4 and MHC class II-deficient mice were poorly protected (14). Despite their importance, little is known about the breadth of VACV-specific CD4 ϩ T cell responses and the nature of the Ags recognized. In contrast, recent studies have defined the Ags and epitopes recognized by CD8 ϩ T cell responses following VACV infection in mice and humans (15-21). The breadth of CD8 ϩ T cell VACV responses is large with a total of 103 Ags recognized thus far in total, most of which are early Ags. CD8 ϩ T cells equally recognize VACV structural proteins, regulatory proteins, and virulence factors.To understand the impact of CD4 ϩ T cell responses and the dynamic development of CD8 ϩ T cell responses, it is important to similarly define the breadth and nature of CD4 ϩ T cell responses.Several studies have shown that the number of CD4 ϩ T cells involved in responses are of lower magnitude when compared with CD8 ϩ T cell responses, both in general and also in the case of VACV infection (22)(23)(24)(25). It is currently unknown whether this lower magnitude is due to the recognition of fewer epitopes, but with each epitope being recognized by responses of similar magnitude. Alternatively, it is possible that the overall lower CD4 ϩ T cell response is reflective of a comparable number of epitopes, each associated with ...