Cytomegaloviral determinants of CD8+ T cell programming and RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) insert-expressing, 68–1 Rhesus Cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex (MHC)-E- and -II-restricted, SIV-specific CD8(+) T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) has not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68–1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/Rh157.4 and Rh158–161), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences (UL128/UL130; UL146/UL147) leads to either of two distinct CD8(+) T cell response types – MHC-Ia-restricted-only, or a mix of MHC-II- and MHC-Ia-restricted CD8(+) T cells. Response magnitude and functional differentiation are similar to RhCMV 68–1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8(+) T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector

Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays.

Highly sensitive detection of HIV-1 nucleic acids is of critical importance for evaluating treatment interventions superimposed on combination antiretroviral therapy (cART) in HIV-1 infected individuals. SIV infection of rhesus macaques models many key aspects of human HIV-1 infection and plays a key role in evaluation of approaches for prevention, treatment and attempted eradication of HIV infection. Here we describe two droplet digital PCR (ddPCR) assays, a ddPCR DNA assay and an RT-ddPCR RNA assay for detecting simian immunodeficiency virus (SIV) on the RainDance platform. We demonstrate that RainDance ddPCR can tolerate significantly higher cell DNA input without inhibition on a per reaction basis (compared to both qPCR and Bio-Rad ddPCR), thus allowing a large quantity of sample to be analyzed in each reaction. In addition, the combination of a high processivity RT enzyme and RainDance ddPCR could overcome inhibition in severely inhibited (99.99% inhibition in qPCR quantification) viral RNA samples. These assays offer valuable tools for assessing low level viral production/replication and strategies for targeting residual virus in the setting of cART suppression of viral replication. The methodologies presented here can be adapted for a broad range of applications where highly sensitive nucleic acid detection is required

Development and optimization of a simian immunodeficiency virus (SIV) droplet digital PCR (ddPCR) assay.

Accurate and sensitive quantification of rebound competent HIV that persists despite combination antiretroviral treatment (cART), including in latently infected cells (i.e., viral reservoir), is critical for evaluating cure strategies for decreasing or eliminating this reservoir. Simian immunodeficiency virus (SIV)-infected Rhesus macaques are an important non-human primate (NHP) system for studying potential cure strategies as they model many key aspects of human HIV-infection including the persistence of a latent viral reservoir in resting memory CD4+ T cells in animals receiving prolonged cART. In this report, we describe the design and testing of a sensitive SIV droplet digital PCR (ddPCR) assay through exploring the combination and optimization of different probe systems (including single, double quencher probes and minor groove binder (MGB) probes) and reaction conditions to eliminate background signal(s), ensure distinct target signal cluster separation from non-target signals, and enable detection and quantification of low level authentic target signals. Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in a large background of nucleic acid input derived from cell or tissue sources