Open Reading Frames Carried on UL/b′ Are Implicated in Shedding and Horizontal Transmission of Rhesus Cytomegalovirus in Rhesus Monkeys

Abstract

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a fulllength UL/b region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one naïve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus with a worldwide seroprevalence of 50 to Ͼ90% in adults (8,14). HCMV elicits a subclinical outcome for the majority of infections in immunocompetent individuals but is a significant pathogen in those without a fully functional immune system. Groups at increased risk for HCMV clinical sequelae include immunologically immature fetuses, immunosuppressed transplant recipients, and immunodeficient AIDS patients. The nearly 4-decade quest for a vaccine that confers protective immunity against HCMV infection has focused primarily on inhibiting primary infection in women without preconceptional immunity to HCMV to prevent transplacental transmission of the virus to the fetus (30). Progress on this front has been reported for a subunit vaccine directed at viral glycoprotein B (gB), which is essential for virus attachment to fibroblasts and is a prominent target of neutralizing antibodies during natural infection (38). A clinical trial with seronegative pregnant women demonstrated that vaccination against gB can reduce primary infection in the vaccine recipients by 50% compared to levels of infection in unvaccinated controls (49). The absence of complete protection in the vaccinees suggests that...

Preexisting antibodies can protect against congenital cytomegalovirus infection in monkeys

“…challenge with a mixture of RhCMV strains 180.92 (originally isolated from the lung of an immune-compromised rhesus macaque) (30) and UCD52/59 (originally isolated from urine of seropositive monkeys) (31). Each monkey was administered 2 × 10 6 50% tissue culture infective dose (TCID 50 ) RhCMV 180.92 in one arm and 1 × 10 6 TCID 50 each of RhCMV UCD52 and UCD59 in the other arm, diluted in serum-free RPMI.…”

Vaccination against a Virus-Encoded Cytokine Significantly Restricts Viral Challenge

“…Four RhCMV-negative rhesus macaques were immunized with a combination of rhcmvIL-10M1 and rhcmvIL-10M2 using a heterologous DNA prime-protein boost immunization strategy (23 (23). The four immunized macaques and four mock-immunized RhCMV-uninfected controls were then challenged subcutaneously with the epitheliotropic RhCMV strain UCD59 at 1,000 PFU (24).…”

“…Oral swabs, urine, and blood were collected from anesthetized animals and processed according to our published procedures (24,25). Saliva was collected via oral swabs of the buccal pouch and subsequently mixed with 2 ml of PBS.…”

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains

“…Accordingly, the observed differences between 180.92 infection in MKE and RRPE could be related to the differences in preparation of 180.92 stocks (Telo-RF-propagated vs MKE-propagated) and tissue origins of EP (retina vs kidney). Nevertheless, it should be mentioned that the growth efficiency of RhCMV strains in MKE in vitro is commensurate to their in vivo shedding profiles (magnitude and frequency) after infection of macaques (Assaf et al, 2014;Oxford et al, 2011), suggesting the relevance of MKE in RhCMV natural transmission.…”

“…UCD52 and UCD59, containing full- length UL/b¢ regions (GenBank accession no. GU552456 and EU130540, respectively), were isolates from the urine of SIV-infected monkeys and were passaged unknown times in MRC-5 and WI-38 and then passaged three times in RhDF (Oxford et al, 2011). RhCMV 180.92 was obtained from Dr Amitinder Kaur (New England Primate Research Center) after 13 passages in MRC-5 and primary rhesus macaque fibroblasts (Rivailler et al, 2006), and subsequently passaged twice in Telo-RF in our laboratory.…”