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

Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68-1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68-1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68-1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68-1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68-1 and 68-1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics

Nucleotide sequence variation across RhCMV and CyCMV.

<p>A) Overall nucleotide diversity within either RhCMV isolates, CyCMV isolates, or MCM CyCMV isolates. B) Graphs indicate the nucleotide diversity across the genome, calculated using overlapping 100 bp sliding windows, incrementing by 50 bp per window. C) Amino acid alignments showing changes in UL128, UL130 and UL131a for each strain when compared to the consensus sequence. D) Amino acid alignments showing changes in gB, or E) gO and gH for each strain when compared against the consensus sequence. Colored dashes represent amino acid differences from the CMV consensus sequence.</p

Gene map of laboratory strains of RhCMV.

<p>Gene map shows UL36, UL128, and UL130,genes as expressed by different laboratory strains of RhCMV. Each box represents a distinct exon.</p

RhCMV replicates on primary MCM fibroblasts <i>in vitro</i> and compromises MHC-I presentation of SIV transgene peptides.

<p>A) Multi-step growth curves reveal the ability of RhCMV 68–1 and RhCMV 68–1.2 to replicate on primary RM and MCM fibroblasts (PFU = plaque forming units). B) ELISpot shows IFNγ secretion by SIV-specific CD8+ T cell lines derived from RM and MCM following stimulation with RhCMV/SIV vector-infected, MHC-matched primary fibroblasts (compared to IFNγ secretion following stimulation with peptide-pulsed primary fibroblasts).</p

RhCMV vectors based on expression of UL36, UL128, UL130, and the Pentameric Complex (PC).

<p>RhCMV vectors based on expression of UL36, UL128, UL130, and the Pentameric Complex (PC).</p

RhCMV 68–1.2 infects MCM.

<p>A) Study timeline showing RhCMV challenges and urine collecting in MCM. B) <i>Ex vivo</i> T cell responses in blood following RhCMV challenge. Measured as the frequency of IFNγ+TNFα+ T cells following stimulation with peptides spanning SIV protein ORFs. Corrected for frequency within memory T cell compartment. C) Absolute CD8+ cell counts in peripheral blood from four MCM following administration of CD8-depleting antibody. D) <i>Ex vivo</i> CD4+ T cell responses in BAL following RhCMV challenge. E) Western blots show shedding of RhCMV/SIV 68–1.2 vectors, but not RhCMV/SIV 68–1 vectors, in the urine of immunocompromised MCM.</p

Coding potential of RhCMV and CyCMV strains.

<p>Full genomes of our viral isolates were assembled and aligned to each other to compare the predicted viral coding regions. Start and stop codons were determined by comparison of all published RhCMV and CyCMV genomes and the predicted full genome coding content of RhCMV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006014#ppat.1006014.ref011" target="_blank">11</a>] was used to determine maximal low passage viral coding potential. Hypothetical full length strain 68–1 with all lab adaptations repaired was included as a theoretical full length isolate [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006014#ppat.1006014.ref025" target="_blank">25</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006014#ppat.1006014.ref029" target="_blank">29</a>]. ORFs in our isolates showing frame shift mutations or point mutations leading to the usage of a premature stop codons compared to the majority predicted stop codon leading to shortened ORFs are indicated in blue, whereas ORF containing frameshift leading to elongations compared to the predicted majority sequence are indicated in green. ORFs that are fully or mostly missing in our isolates compared to and the predicted full genome coding content of RhCMV are highlighted in red.</p

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques

<div><p>Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68–1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells <i>in vitro</i>, RhCMV 68–1 failed to productively infect MCM <i>in vivo</i>, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68–1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68–1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68–1 and 68–1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.</p></div