N5-1

We have demonstrated that the mouse chemokine N51, also known as KC, can compete for 125I-human interleukin-8 (IL-8) binding to NIH 3T3 cells expressing the human IL-8 receptor beta (NIH-IL-8R beta) but not the IL-8 receptor alpha (NIH-IL-8R alpha). In addition, we used the chimeras between N51 and IL-8 described previously (Heinrich, J. N., O'Rourke, E. C., Chen, L., Gray, H., Dorfman, K. S., and Bravo, R. (1994) Mol. Cell. Biol. 14, 2849-2861; Heinrich, J. N., and Bravo, R. (1995) J. Biol. Chem. 270, 4987-4989) to evaluate possible contributions of equivalent domains from each chemokine to binding and specificity. Specifically, the amino acid sequences between cysteines 2 and 3 or between cysteines 3 and 4 or the alpha-helical C-terminal end (domains I, II, and III, respectively) of one of the chemokines was exchanged with the corresponding sequence of the other and vice versa. Chimeras of IL-8 containing either domain II or III of N51 behaved similarly, but not identically, to IL-8 in competing 125I-IL-8 binding with both NIH-IL-8R alpha cells and NIH-IL-8R beta cells. The IL-8 chimera containing domain I of N51 did not compete. On the other hand, N51 competes 125I-IL-8 binding with NIH-IL-8R beta but not NIH-IL-8R alpha cells. The N51 chimera containing domain I of IL-8 was an agonist with NIH-IL-8R alpha cells and was an even more potent agonist with NIH-IL-8R beta cells. On the latter cells it was more potent than either IL-8 or N51. The N51 chimera containing domain II of IL-8, compared with N51, was a partial agonist with NIH-IL-8R alpha cells but was equivalent to N51 with NIH-IL-8R beta cells. However, N51 chimera containing domain III of IL-8 was a partial agonist with both cells. The results are consistent with the observations we originally made with human neutrophils and the NIH mouse IL-8R beta cells, i.e. domain I confers binding specificity for IL-8 and domains II and III of IL-8 and N51 may be interchangeable but they are not equivalent. Although we originally hypothesize that domains II and III confer binding specificity to N51, these results emphasize the role of domain III.

The exploitation of various human immunodeficiency virus type-1 (HIV-1) vaccines has posed great challenges for the researchers in precisely evaluating the vaccine-induced immune responses, however, the understanding of vaccination response suffers from the lack of unbiased characterization of the immune landscape. The rapid development of high throughput sequencing (HTS) makes it possible to scrutinize the extremely complicated immunological responses during vaccination. In the current study, three vaccines, namely N36, N51, and 5-Helix based on the HIV-1 gp41 pre-hairpin fusion intermediate were applied in rhesus macaques. We assessed the longitudinal vaccine responses using HTS, which delineated the evolutionary features of both T cell and B cell receptor repertoires with extreme diversities. Upon vaccination, we unexpectedly found significant discrepancies in the landscapes of T-cell and B-cell repertoires, together with the detection of significant class switching and the lineage expansion of the B cell receptor or immunoglobulin heavy chain (IGH) repertoire. The vaccine-induced expansions of lineages were further evaluated for mutation rate, lineage abundance, and lineage size features in their IGH repertoires. Collectively, these findings conclude that the N51 vaccine displayed superior performance in inducing the class-switch of B cell isotypes and promoting mutations of IgM B cells. In addition, the systematic HTS analysis of the immune repertoires demonstrates its wide applicability in enhancing the understanding of immunologic changes during pathogen challenge, and will guide the development, evaluation, and exploitation of new generation of diagnostic markers, immunotherapies, and vaccine strategies.