Long-term sterile immunity induced by an adjuvant-containing live-attenuated AIDS virus

Antigen 85B (Ag85B) is one of the most dominant proteins secreted from most mycobacterial species, and it induces Th1-type immune responses as an adjuvant. We genetically constructed a live attenuated simian human immunodeficiency virus to express the adjuvant molecule Ag85B (SHIV-Ag85B). SHIV-Ag85B could not be detected 4 weeks after injection in cynomolgus macaques, and strong SHIV-specific T cell responses were induced in these macaques. When these macaques in which SHIV-Ag85B had become undetectable were challenged with pathogenic SHIV89.6P at 37 weeks after SHIV-Ag85B became undetectable, SHIV89.6P could not be detected after the challenge. Eradication of SHIV89.6P was confirmed by adoptive transfer experiments and CD8-depletion studies. The SHIV-Ag85B-inoculated macaques showed enhancement of Gag-specific monofunctional and polyfunctional CD8+ T cells in the acute phase of pathogenic SHIV challenge. The results suggest that SHIV-Ag85B elicited strong sterile immune responses against pathogenic SHIV and that it may lead to the development of a vaccine for AIDS virus infection. Importance Development of an effective HIV vaccine has been a major priority to control the worldwide AIDS epidemic. The moderately attenuated prototypic vaccine strain SIVmac239Δnef has been used in various studies; however, it does not provide sufficient effects to prevent infection. The use of adjuvant in vaccination is thought to be useful for enhancing the immune responses to various pathogens. In the present study, we constructed a live attenuated SHIV virus expressing adjuvant molecule Ag85B and assessed vaccine effects in cynomolgus macaques. The present study shows that live-attenuated SHIV expressing Ag85B elicits viral antigen-specific polyfunctional CD8+ T cell responses against pathogenic SHIV and provide the possibility of eradicating a pathogenic lentivirus from infected animals.

3 Introduction (Fig. 3A). These macaques showed very low CD4 + T cell counts (< 100 cells/μl) during 217 the observation period (Fig. 3B) and showed AIDS symptoms at 402 to 1225 days after 218 the challenge.  #500245 and #502833) showed stable viremia above 10 4 to 10 7 copies/ml of viral RNA 250 ( Fig. 3A and 4A). We next analyzed SHIV antigen-specific production of IFN-γ by 251 ELISPOT assays in these experimental macaques after the challenge. In six 252 SHIV-Ag85B-undetected macaques, ELISPOT responses were increased dramatically at 253 2 weeks after the challenge (Fig. 5A). The responses were reduced in most of those 254 macaques at 12 weeks after the challenge. In only one of the SHIV-Ag85B-undetected 255 macaques, the IFN-γ ELISPOT response was moderately increased at 2 weeks after the 256 challenge and was maintained at 12 weeks after the challenge. The response was similar 257 to ELISPOT responses of SHIV-NI. The control macaques showed weak  ELISPOT responses at all time points (Fig. 5A). The polyfunctionality of 259 Gag/pol-specific T cells was also examined by flow cytometry. On the day of the 260 challenge, we did not detect polyfunctional Gag/pol-specific CD4 + and CD8 + T cells in 261 any of the macaques (Fig. 5B and Fig. S6). In six SHIV-Ag85B-undetected macaques, 262 there was preferential accumulation of Gag/pol-specific, triple-positive (IFN-γ, TNF-α 263 and IL2), double-positive (IFN-γ and TNF-α), and IFN-γ or TNF-α-single positive 264 CD8 + T cells at 2 weeks after the challenge (Fig. 5B). In addition, the percentages of 265 these polyfunctional Gag/pol-specific CD8 + T cells in six SHIV-Ag85B-undetected 266 macaques were higher than those in SHIV-NI-infected macaques and control macaques 267 (Fig. 5B). We found polyfunctional Gag/pol-specific CD4 + T cells in six 268 SHIV-Ag85B-undetected macaques, but the percentages were similar to those in 269 SHIV-NI-infected macaques at 2 weeks after the challenge (Fig. S6). We also analyzed 270 the memory phenotype of SHIV antigen-specific CD8 + T cells in those three groups.

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After the pathogenic SHIV89.6P challenge, we measured SHIV-specific antibody in

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To examine virus clearance in the animals, proviral DNAs in various lymphoid    intravenously with heterologous pathogenic SHIV89.6P at 37 weeks after inoculation.

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The design of the macaque study is outlined in Fig.1A.  The levels of SHIV infection were monitored by measuring plasma viral RNA loads 571 using highly sensitive quantitative real-time RT-PCR as described previously (37,48).

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Briefly, viral RNA was isolated from plasma using a MagNA PureCompact Nucleic  626 Antigen-specific cellular immune responses were assessed in multiparameter 627 intracellular cytokine staining (ICS) assays as described previously (50,51). Briefly,  Data are represented as means ± SD. A P value of <0.05 is considered significant.

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Statistical analyses were performed using the Mann-Whitney U test. Statistically 674 significant differences compared with the control are indicated by asterisks.  The authors declare no competing interests.   Magnification×200. Black scale bars, 100 μm.