Simian varicella virus infection of rhesus macaques recapitulates essential features of varicella zoster virus infection in humans

Abstract

Simian varicella virus (SVV), the etiologic agent of naturally occurring varicella in primates, is genetically and antigenically closely related to human varicella zoster virus (VZV). Early attempts to develop a model of VZV pathogenesis and latency in nonhuman primates (NHP) resulted in persistent infection. More recent models successfully produced latency; however, only a minority of monkeys became viremic and seroconverted. Thus, previous NHP models were not ideally suited to analyze the immune response to SVV during acute infection and the transition to latency. Here, we show for the first time that intrabronchial inoculation of rhesus macaques with SVV closely mimics naturally occurring varicella (chickenpox) in humans. Infected monkeys developed varicella and viremia that resolved 21 days after infection. Months later, viral DNA was detected only in ganglia and not in non-ganglionic tissues. Like VZV latency in human ganglia, transcripts corresponding to SVV ORFs 21, 62, 63 and 66, but not ORF 40, were detected by RT-PCR. In addition, as described for VZV, SVV ORF 63 protein was detected in the cytoplasm of neurons in latently infected monkey ganglia by immunohistochemistry. We also present the first in depth analysis of the immune response to SVV. Infected animals produced a strong humoral and cell-mediated immune response to SVV, as assessed by immunohistology, serology and flow cytometry. Intrabronchial inoculation of rhesus macaques with SVV provides a novel model to analyze viral and immunological mechanisms of VZV latency and reactivation.

Figure 1. Varicella and the presence of SVV DNA and viral antigen in rhesus macaques inoculated intrabronchially with SVV.

(A) Varicella rash on skin of monkey 24953 9 days after intrabronchial infection. (B) Detection of SVV DNA by real-time PCR in punch biopsies of skin from all 4 monkeys during varicella. (C) Immunohistochemical detection of SVV antigen with antibodies directed against SVV glycoproteins H and L in skin of monkey 24953 during varicella, with staining in superficial and deep layers of skin. (D) Skin staining of monkey 24953 with normal rabbit serum. (E) SVV DNA levels in peripheral blood mononuclear cells (PBMC) and samples of all monkeys as assessed by quantitative real-time PCR. SVV DNA was first detected 7 days post-infection (dpi), peaked at 10 dpi and was not detected 21 dpi. (F) SVV DNA levels in bronchial alveolar lavage (BAL) samples in all four monkeys as assessed by quantitative real-time PCR. SVV was first detected in BAL as early as 3 dpi, peaked 3–7 dpi and was not detected 17 dpi.

Figure 2. Detection of SVV ORF 63 protein in the cytoplasm of neurons in monkey ganglia latently infected with SVV.

Paraformaldehyde-fixed, paraffin-embedded sections (5 µ) of thoracic (th) ganglia from monkey 23986, trigeminal (tg) ganglia from monkey 23942 and thoracic ganglia from monkey 24953 were analyzed by immunohistochemistry using a 1∶800 dilution of rabbit anti-VZV ORF 63 (anti-VZV63), a 1∶1000 dilution of rabbit anti-VZV ORF 61 (anti VZV61) antiserum or normal rabbit serum (NRS). SVV ORF 63 protein was detected in thoracic and trigeminal ganglia of monkeys 23986 and 23942, respectively, and SVV ORF 61 protein was detected in thoracic ganglia of monkey 24953. No SVV antigen was seen using normal rabbit serum. SVV ORF63 protein, but not SVV ORF61 protein was detected in trigeminal ganglia of monkey 23942.

Figure 3. SVV infection of rhesus macaques elicits a B cell response after the appearance of varicella rash.

B cells were separated into three subsets based on expression of IgD and CD27: CD27−IgD+; CD27+IgD+; and isotype-switched CD27+IgD− cells. A representative profile from PBMCs obtained from monkey 24953 on day 0 (A, left) is shown. B cell proliferation within CD27+IgD+ and CD27+IgD− subsets was measured using flow cytometry based on expression of the nuclear protein Ki67, which is up-regulated upon entry into the cell cycle. B cell proliferation was minimal before SVV infection (0 dpi, A, middle), but increased dramatically 14 dpi (A, right). Throughout the course of SVV infection in all 4 monkeys, the number of Ki67+ CD27+IgD+ (panel B) and CD27+IgD− B cells (C) increased dramatically 14 dpi compared to 0 dpi in PBMCs. In BAL of all 4 monkeys, increased numbers of Ki67+ IgD−CD27+ B cells were seen throughout the course of infection compared to 0 dpi (panel D), although no significant proliferation of IgD+CD27+ B cells was detected (data not shown). In all 4 monkeys, SVV-specific IgG antibodies appeared 7 dpi, peaked 18–21 dpi (as detected by ELISA) and remained stable up to 70 dpi (E).

Figure 4. SVV infection of rhesus macaques elicits a robust peripheral T cell response.

CD4 and CD8 T cells were separated into three subsets based on expression of CD28 and CD95: naïve (CD28+CD95−); central memory (CM, CD28+CD95+); and effector memory (EM, CD28−CD95+) cells. A representative profile of CD8 T cell subsets of monkey 24953 on 0 dpi is shown (A, left). T cell proliferation was measured as described in Figure 3 based on expression of the nuclear protein Ki67 using flow cytometry. T cell proliferation dramatically increased 14 dpi (A, right) compared to 0 dpi (A, middle). Fold increases (relative to 0 dpi) in the numbers of Ki67+ CD4 CM and EM (B and C) and CD8 CM and EM T cells (D and E) in PBMCs indicate a peak proliferative response of all T cell subsets 14 dpi in all monkeys (B–E).

Figure 5. SVV-induced T cell proliferation occurs earlier in BAL than in peripheral blood.

In contrast to PBMCs, BAL revealed only CM and EM T cells subsets as illustrated by a profile of CD8 T cells from 24953 day 0 (A, left). T cell proliferation was measured as described in Figure 3. A representative example of Ki67 staining within CD8 EM T cells from monkey 24953 on 0 dpi (A, middle) and 14 dpi (A, right) is shown. Fold increases (relative to 0 dpi) in the percentages of Ki67+ CD4/CM, CD4/EM, CD8/CM and CD8/EM T cells subsets in BAL from all 4 monkeys indicate a robust proliferative response of all T cell subsets 7–14 dpi in all monkeys (B–E).

Figure 6. Appearance of SVV-specific CD4 T cells correlates with Ki67 expression.

The frequency of SVV-specific T cells in PBMCs and BAL was measured by intracellular cytokine staining. (A) Representative examples of IFNγ and TNFα production by CD4 CM, CD4 EM, CD8 CM and CD8 EM T cells from BAL of monkey 24953 7 dpi after stimulation with vaccinia virus (VV) lysate (negative control) and SVV lysate. A robust response by CD4 CM and CD4 EM cells was detected after exposure to SVV lysates, but not VV lysates (A, left), whereas no response by CD8 CM and CD8 EM cells was seen after exposure to either SVV or VV lysate (A, right). The percentage of responding (IFNγ+ alone or both IFNγ+ and TNFα+) CD4 CM and CD4 EM T cells in BAL (B and C) and PBMCs (D and E) in all 4 monkeys was determined and averaged. At all time points, more SVV-responsive CD4/CM and CD4/EM cells were found in BAL (B and C) than in PBMCs (D and E).

Figure 7. Increased granzyme B expression in EM T cells after SVV infection.

(A) Intracellular granzyme B expression in CD4 and CD8 T cell subsets as assessed by flow cytometry. An example of CD8 T cell subsets in BAL on day 0 from animal 24953 is shown in left panel. Frequency of granzyme B+ EM T cells increased dramatically after SVV infection as shown in BAL-resident CD8 EM T cells from monkey 24953 (A, middle and right), while the increase in granzyme B+ CM T cells was comparatively modest (data not shown). Measurement of fold increases (compared to 0 dpi) in the percentages of granzyme B-expressing CD8/EM and CD4/EM from BAL and PBMCs during the course of infection indicated a significant increase in these percentages in both BAL and PBMCs 12–14 dpi in all samples (B–E).