Expression of CD40L by the ALVAC-SIV vector abrogates T cell responses in 1 macaques 2

38 Immunization with recombinant ALVAC/gp120 alum vaccine provided modest 39 protection from HIV-1 and SIV acquisition in humans and macaques. Vaccine-mediated 40 protection was associated with the elicitation of IgG against the envelope V2 loop and of 41 envelope-specific CD4 + T cell responses. We hypothesized that the simultaneous 42 expression of the co-stimulatory molecule CD40L (CD154) by the ALVAC-HIV vector 43 could increase both protective humoral and cellular responses. We engineered an 44 ALVAC-SIV co-expressing CD40L with SIV mac251 (ALVAC-SIV/CD40L) gag , pol , and 45 env genes. We compared its immunogenicity in macaques with a canonical ALVAC-SIV, 46 with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC- 47 SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 48 neutralizing antibodies against the gp120. The increase in humoral responses was 49 associated with the expression of the membrane-bound form of the CD40L by CD4 + T 50 cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting 51 effect on CD4 + Th1 helper responses, and instead favored the induction of myeloid 52 derived suppressor cells, the immune-suppressive IL-10 cytokine, and the down- 53 modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques 54 from SIV acquisition. Taken together, these results underlie the importance of balanced 55 vaccine-induced activating versus suppressive immune responses in affording protection 56 from HIV. CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective 62 cytotoxic and humoral responses against pathogens. Because of its immunomodulatory 63 function, CD40L has been used to enhance immune responses to vaccines, including 64 candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a 65 strategy combining canarypox virus-based vector (ALVAC) together with an envelope 66 protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing 67 HIV-1/SIV acquisition in humans and macaques. In both species, protection was 68 associated with vaccine-induced antibodies against the HIV envelope and CD4 + T cell 69 responses, including Type 1 antiviral responses. In this study, we tested whether 70 augmenting CD40L expression by co-expressing it with the ALVAC vector could 71 increase the protective immune responses. Although co-expression of CD40L did 72 increase humoral responses, it blunted Type 1 CD4 + T cells responses against the SIV 73 envelope protein, and failed to protect macaques from viral infection.