Late gene expression-deficient cytomegalovirus vectors elicit conventional T cells that do not protect against SIV

Rhesus cytomegalovirus-based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory-based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71). Here, we examined the impact of an even more stringent attenuation strategy on vector-induced immune protection against SIV. Fusion of the FK506-binding protein (FKBP) degradation domain to Rh108, the orthologue of the essential human CMV (HCMV) late gene transcription factor UL79, generated RhCMV/SIV vectors that conditionally replicate only when the FK506 analog Shield-1 is present. Despite lacking in vivo dissemination and reduced innate and B cell responses to vaccination, Rh108-deficient 68-1 RhCMV/SIV vectors elicited high-frequency, durable, EM-biased, SIV-specific T cell responses in RhCMV-seropositive RMs at doses of ≥ 1 × 106 PFU. Strikingly, elicited CD8+ T cells exclusively targeted MHC-Ia-restricted epitopes and failed to protect against SIVmac239 challenge. Thus, Rh108-dependent late gene expression is required for both induction of MHC-E-restricted T cells and protection against SIV

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques

Interleukin-15 response signature predicts RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in similar to 55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8(+) T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8(+) T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8(+) T cells to mediate protection against SIV challenge