Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine

Abstract

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8 + T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8 + and CD4 + T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4 + T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8 + and CD4 + T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods -also demonstrated in principle for vaccinia virus for both CD8 + and CD4 + T cells -should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens. IntroductionHerpes simplex virus type 1 (HSV-1) infects 60% of the US population and has a significant cumulative health care burden in addition to causing painful recurrent oral-labial infections. For example, brain and eye infections can cause permanent damage or blindness (1). HSV-1 also causes approximately 50% of clinical first-episode genital herpes in the United States. Vaccines for HSV that have been tested thus far have failed in clinical trials, including a recent phase III trial of an adjuvanted glycoprotein D (gD2) product (2). This vaccine elicits antibody and CD4 + T cell responses but fails to induce CD8 responses. Newer platforms can elicit CD8 + and CD4 + cells, but they require rationally selected T cell antigens. We therefore developed methods to permit measurement of both CD8 and CD4 responses to the complete HSV-1 proteome to begin rational prioritization of next-generation vaccine candidates.Several recent observations support the concept that an effective HSV vaccine will need to induce coordinated CD8 + and CD4 + T cell responses. HSV-1-specific CD8 + T cells localize to the site of HSV-1 infection in human and murine trigeminal ganglia (TG) (3-5), and both HSV-specific CD8 + and CD4 + T cells localize to acute and healed sites of skin infection in mice and humans, sug-

Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection

“…Antigen and epitope mapping. To map the antigen specificity of the CD4 ϩ T cells in genital biopsy specimens, we used the HSV-2 ORFeome, a tool which can be used to study proliferative responses to all HSV-2 open reading frames and which allows dissection of antigen-specific T cell responses (21). We observed reactivity to several HSV-2 antigens (range, 3 to 18) in lesion biopsy specimens in three of three participants who had HSV-specific cells detected from genital biopsy specimens (see Table S2 in the supplemental material).…”

“…Autologous peripheral blood mononuclear cells (PBMCs) labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) were used as antigen-presenting cells (APC) at a 1:1 ratio with expanded bulk skin lymphocyte responders as previously described (20). Brefeldin A was added at 1 h, cells were labeled for CD3 and CD4, and intracellular gamma interferon (IFN-␥), tumor necrosis factor alpha (TNF-␣), and interleukin-2 (IL-2) were measured at 6 h as previously described (21). A FACSantoflow cytometer (BD Biosciences) with FlowJo software (Tree Star) was used for determinations performed by gating on lymphocyte forward and side scatter, excluding CFSE-positive cells.…”

“…For biopsy specimens from participants who had HSV-specific CD4 ϩ T cells, we used a complete CD4 ϩ T cell HSV-2 protein set, termed the HSV-2 open reading frame (ORF)eome, to test for proliferative responses to individual HSV proteins, as described for HSV-1 (21). Briefly, bulk expanded CD4 ϩ lymphocytes, autologous irradiated PBMCs used as APC, and recombinant HSV-2 proteins were plated in duplicate in 96-well U-bottom plates.…”

“…[ 3 H]thymidine incorporation was measured after 3 days to detect cell proliferation. The criterion for positivity for each antigen was set as the mean plus 2.1 times the standard deviation of the [ 3 H]thymidine incorporation for 54 negativecontrol antigens (21). In one participant tested with both the HSV-1 and HSV-2 ORFeomes, CD4 ϩ cells in both lesion and asymptomatic biopsy specimens showed reactivity to VP16.…”

Extensive CD4 and CD8 T Cell Cross-Reactivity between Alphaherpesviruses

“…Some were studied previously (Supplemental Table I). HSV-1 immunity was studied in participants 21 and 23 (25, 27), zoster vaccine immunity in participants 1, 2, 3, 4, and 26 (16), T-cell responses to HSV-2 in subjects 30-34 (15), and drug reactions in subject 35 (28). Subjects 6-19 and 26-29 were genetically confirmed HSV-1-infected monozygotic twins (29, 30), with consecutively numbers denoting twin pairs.…”