The T cell coreceptor CD4 is a transmembrane glycoprotein belonging to the Ig superfamily and is essential for cell-mediated immunity. Two different genes were identified in rainbow trout that resemble mammalian CD4. One (trout CD4) encodes four extracellular Ig domains reminiscent of mammalian CD4, whereas the other (CD4REL) codes for two Ig domains. Structural motifs within the amino acid sequences suggest that the two Ig domains of CD4REL duplicated to generate the four-domain molecule of CD4 and the related gene, lymphocyte activation gene-3. Here we present evidence that both of these molecules in trout are homologous to mammalian CD4 and that teleosts encode an additional CD4 family member, lymphocyte activation gene-3, which is a marker for activated T cells. The syntenic relationships of similar genes in other teleost and non-fish genomes provide evidence for the likely evolution of CD4-related molecules in vertebrates, with CD4REL likely representing the primordial form in fish. Expression of both CD4 genes is highest in the thymus and spleen, and mRNA expression of these genes is limited to surface IgM− lymphocytes. consistent with a role for T cell functionality. Finally, the intracellular regions of both CD4 and CD4REL possess the canonical CXC motif involved in the interaction of CD4 with p56LCK, implying that similar mechanisms for CD4+ T cell activation are present in all vertebrates. Our results therefore raise new questions about T cell development and functionality in lower vertebrates that cannot be answered by current mammalian models and, thus, is of fundamental importance for understanding the evolution of cell-mediated immunity in gnathosomes.

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-

Summary Expression of inducible nitric oxide synthase (iNOS) mRNA was detected in a recently developed goldfish macrophage cell line by RT-PCR. using degenerate primers designed against conserved nucleotide motifs within the different mammalian isoforms of NOS. Increased expression of iNOS poststimulation with LPS was found, and suggests that it is a functional enzyme in goldfish macrophages. supporting the view that iNOS regulation is prctranslational. The nucleotide sequence translated in one reading frame with no stop codons to produce a partial peptide containing 164 amino acids, with highest homology (85%) to a recently identified rainbow trout iNOS sequence. The pcplidc translation also gave an insight into the conservation of binding motifs, since two cofactor binding sites were present in the amplified PCR product (FMN and calmodulin). In addition, a 42 aa motif present in the region just upstream of the FMN binding motif of mammalian endothelial and neuronal NOS isoforms was absent in the translation, in agreement with every published sequence for iNOS. Finally, the translation was used to construct an unrooted phylogenetic tree.

BackgroundWe conducted a phase 1, randomized, double-blind, placebo-controlled trial of a replication-defective HSV-2 vaccine, HSV529 (deleted for UL5 and UL29), in 60 healthy adults aged 18 to 40 years.MethodsSubjects were enrolled in groups of 20 from 3 serogroups: HSV1+ or 
-/HSV2+ (group 1), HSV1+/HSV2– (group 2), and HSV1-/HSV2– (group 3). At months 0, 1, and 6, 15 subjects in each group received HSV529 intramuscularly and 5 subjects received placebo. The primary endpoint was the frequency of solicited injection site and systemic reactions from day 0 to 7 after each vaccination and unsolicited adverse events up to 6 months after the last dose.Results89% of vaccine recipients experienced a mild to moderate solicited injection site reaction vs. 47% of placebo recipients (P = 0.006, 95% CI 0.129, 0.676) that did not preclude additional doses. 64% of vaccine recipients experienced solicited systemic reactions vs. 53% of placebo recipients (P = 0.44, 95% CI -0.179, 0.402). Two serious adverse events occurred in 2 participants and were assessed as unrelated to HSV529 administration. Serum neutralizing antibody titers significantly increased from baseline after 3 doses of HSV529 compared with placebo in group 3 only (P < 0.001). This increase persisted up to 6 months after the third dose of vaccine (P < 0.001). Serum and vaginal antibodies to HSV2 glycoprotein D (gD) also significantly increased after 3 doses of vaccine in group 3 subjects (P < 0.001 and P = 0.012, respectively). The mean vaginal gD titer after 3 doses was about one-third of the mean serum gD titer. In addition, the vaccine induced significant levels of HSV2-specific antibody dependent cellular cytotoxicity (ADCC) after 3 doses in group 3 subjects compared with placebo (P < 0.001). Vaccine-induced CD4 T-cell responses were detected in 46%, 27%, and 36% of subjects in groups 1, 2, and 3, respectively, one month after the third dose of vaccine. CD8 T-cell responses were detected in 8%, 18%, and 14% of subjects in groups 1, 2, and 3, respectively, at the same time point.ConclusionThe HSV529 vaccine was safe, well-tolerated, and immunogenic, eliciting significant neutralizing, gD, and ADCC-mediating antibodies, and modest cellular immune responses in HSV seronegative individuals. NCT01915212Disclosures L. Dropulic, sanofi pasteur: Collaborator, Research support; K. Wang, sanofi pasteur: Collaborator, Research support; M. Oestreich, sanofi pasteur: Collaborator, Research support; H. Pietz, sanofi pasteur: Collaborator, Research support; D. Garabedian, sanofi pasteur: Collaborator, Research support; K. Dowdell, sanofi pasteur: Collaborator, Research support; H. Nguyen, sanofi pasteur: Collaborator, Research support; K. Laing, sanofi pasteur: Research Contractor, payment for conducting T cell assays; D. Koelle, sanofi pasteur: Research Contractor, payment for conducting T cell assays; A. Azose, sanofi pasteur: Research Contractor, Payment for conducting T cell assays; A. Chen, sanofi pasteur: Employee, Salary; L. J. Chang, sanofi pasteur: Employee...