Examining the species-specificity of rhesus macaque cytomegalovirus (RhCMV) in cynomolgus macaques

Abstract

Cytomegalovirus (CMV) is a highly species-specific virus that has co-evolved with its host over millions of years and thus restricting cross-species infection. To examine the extent to which host restriction may prevent cross-species research between closely related non-human primates, we evaluated experimental infection of cynomolgus macaques with a recombinant rhesus macaque-derived CMV (RhCMV-eGFP). Twelve cynomolgus macaques were randomly allocated to three groups: one experimental group (RhCMV-eGFP) and two control groups (UV-inactivated RhCMV-eGFP or media alone). The animals were given two subcutaneous inoculations at week 0 and week 8, and a subset of animals received an intravenous inoculation at week 23. No overt clinical or haematological changes were observed and PBMCs isolated from RhCMV-eGFP inoculated animals had comparable eGFP- and IE-1-specific cellular responses to the control animals. Following inoculation with RhCMV-eGFP, we were unable to detect evidence of infection in any blood or tissue samples up to 4 years post-inoculation, using sensitive viral co-culture, qPCR, and Western blot assays. Co-culture of urine and saliva samples demonstrated the presence of endogenous cynomolgus CMV (CyCMV) cytopathic effect, however no concomitant eGFP expression was observed. The absence of detectable RhCMV-eGFP suggests that the CyCMV-seropositive cynomolgus macaques were not productively infected with RhCMV-eGFP under these inoculation conditions. In a continued effort to develop CMV as a viral vector for an HIV/SIV vaccine, these studies demonstrate that CMV is highly restricted to its host species and can be highly affected by laboratory cell culture. Consideration of the differences between lab-adapted and primary viruses with respect to species range and cell tropism should be a priority in evaluating CMV as vaccine vector for HIV or other pathogens at the preclinical development stage.

Fig 1. Inoculation and sampling schedule.

Twelve cynomolgus macaques were randomly assigned into three groups, RhCMV-eGFP (N = 6), UV-inactivated RhCMV-eGFP control (N = 2), and media control (N = 4). The animals received one subcutaneous inoculation at week 0 with 7x10⁷ PFU of RhCMV-eGFP or UV-inactivated RhCMV-eGFP, or media alone. A subset of animals was subcutaneously boosted at week 8 with 2x10⁷ PFU of RhCMV-eGFP or UV-inactivated RhCMV-eGFP, while the remaining animals received media alone (Table 1). At week 23, one UV-inactivated RhCMV-eGFP control animal (4M) and three media control animals (1M, 2M, 6M) received an intravenous inoculation with RhCMV-eGFP (7x10⁷ PFU). Sample collection schedule is described.

Fig 2. Immunophenotyping.

Absolute values (cells/μl of blood) of T cell subsets (CD3+, CD4+, CD8+, CD4+CD8+), B cells (CD20+) and NK cells (CD16+CD56+) were determined by Flow Cytometry. Each point represents the mean of the animals in the group with error bars showing the standard deviation. The dashed vertical lines (

Fig 3. eGFP protein expression is not detected in RhCMV-eGFP inoculated cynomolgus macaques.

Total protein was isolated from urine co-cultures in Telo-RF infected cells and probed by Western blot analyses with antibodies specific for eGFP (27 kDa), IE (72 kDa), and β-actin (42 kDa) as a lysate control. A) Analyses were performed following the two subcutaneous inoculations (week 16). Animals are listed by animal number as XX. B) In vitro infected Telo-RF cells were used for the eGFP controls with RhCMV-eGFP-infected cells as a positive control (+) and CyCMV_Ott-infected cells as a negative control (-). Western blots from different experiments were combined for analyses.

Fig 4. Anti-eGFP antibody responses present in RhCMV-eGFP and UV-inactivated RhCMV-eGFP control animals.

Anti-eGFP antibody responses were determined by ELISA assay, values represented by optical density (OD). The mean of the replicate wells for each animal (A) or the mean of the animals in each group (B) are shown with error bars denoting the standard deviation. The dashed vertical lines represent subcutaneous inoculations and the solid vertical line represents intravenous RhCMV-eGFP inoculation of four control animals (1M, 2M, 4M, 6M). B) The mean from the animals in each group that were omitted from the second subcutaneous boost at week 8 (RhCMV-eGFP group) or the intravenous inoculation at week 23 (UV-inactivated group) (Table 1) are plotted with an inverted arrow (◉).

Fig 5. Sequence alignment comparing RhCMV-eGFP to RhCMV 68–1 BAC.

Sequence of RhCMV-eGFP obtained from NGS analysis was aligned with that of RhCMV 68–1 BAC (JQ795930) using Geneious Pro 6.1.4 software. Whole genome comparisons yielded 96.2% similarity and the major difference can be attributed to the BAC cassette present in RhCMV68-1 BAC. (∎) refers to eGFP reporter * sequencing error;** 22 bp deletion; *** 56 bp insertion.