An extremely diverse CD4 response to vaccinia virus in humans is revealed by proteome-wide T-cell profiling.

CD4 T cells are required for the maintenance and recall of antiviral CD8 T cells and for antibody responses. Little is known concerning the overall architecture of the CD4 response to complex microbial pathogens. In a whole-proteome approach, 180 predicted open reading frames (ORFs) in the vaccinia virus genome were expressed and tested using responder cells from 20 blood samples from 11 vaccinees. Validation assays established the sensitivity and specificity of the system. Overall, CD4 responses were detected for 122 ORFs (68%). A mean of 39 ORFs were recognized per person (range, 13 to 63). The most frequently recognized ORFS were present in virions, including A3L and A10L (core proteins), WR148 (a fragmented homolog of an orthopoxvirus protein that forms inclusions in cells), H3L (a membrane protein), D13L (a membrane scaffold protein), and L4R (a nucleic acid binding protein). Serum immunoglobulin G profiling by proteome microarray detected responses to 45 (25%) of the ORFs and confirmed recent studies showing a diverse response directed to membrane and nonmembrane antigens. Our results provide the first empirical whole-proteome data set regarding the global CD4 response to full-length proteins in a complex virus and are consistent with the theory that abundant structural proteins are immunodominant.

Dominance and diversity in the primary human CD4 T cell response to replication-competent vaccinia virus.

Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication-incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relationship between Ab and CD4 T cell specificity for complex pathogens.

Diversity in the acute CD8 T cell response to vaccinia virus in humans.

Orthopoxviruses have complex proteomes. Infection provokes a brisk CD8 response, which is required in some systems for recovery from primary infection. Little is known concerning the Ags and epitopes recognized by CD8 T cells. We examined the fine specificity of cloned and bulk human vaccinia-specific CD8 CTL by expressing polypeptide fragments from a library of vaccinia genomic DNA. This epitope discovery method emphasizes virus-specific biological activity, as the responder cells are all reactive with whole vaccinia virus. Sixteen novel epitopes, restricted by several HLA A and B alleles, were defined to the nomamer peptide level in diverse vaccinia open reading frames. An additional seven epitope were mapped to short regions of vaccinia proteins. Targets of the CD8 response included proteins assigned to structural, enzymatic, transcription factor, and immune evasion functions, and included members of all viral kinetic classes. Most epitopes were conserved in other orthopoxviruses. Responses to at least 18 epitopes were detected within a single blood sample, revealing a surprising degree of diversity. These epitopes will be useful in natural history studies of CD8 responses to vaccinia, a nonpersisting virus with long-term memory, and in the design and evaluation of attenuated and replication-incompetent vaccinia strains being tested for variola and monkeypox prevention and for the delivery of heterologous Ags.