Experimental infection of Cynomolgus Macaques (Macaca fascicularis) with human varicella-zoster virus

Abstract

Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family and the causative agent of chickenpox and shingles. To determine the utility of cynomolgus macaques (Macaca fascicularis) as a nonhuman primate model to evaluate VZV-based simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) vaccines, we experimentally inoculated 10 animals with the parental Oka (Oka-P) strain of VZV derived from MeWo or Telo-RF cells. VZV DNA could be detected in the lungs as late as 4 days postinfection, with replicating virus detected by shell vial culture assay in one case. Infection did not result in any overt clinical symptoms but was characterized by humoral and cell-mediated immunity in a time frame and at a magnitude similar to those observed following VZV vaccination in humans. The cell line source of VZV inoculum influenced both the magnitude and polyfunctionality of cell-mediated immunity. Animals mounted a vigorous anamnestic antibody response following a second inoculation 12 weeks later. Inoculations resulted in transient increases in CD4(+) T-cell activation and proliferation, as well as a sustained increase in CD4(+) T cells coexpressing CCR5 and α4β7 integrin. In contrast to previous failed attempts to successfully utilize attenuated VZV-Oka as an SIV vaccine vector in rhesus macaques due to suboptimal infectivity and cellular immunogenicity, the ability to infect cynomolgus macaques with Oka-P VZV should provide a valuable tool for evaluating VZV-vectored SIV/HIV vaccines.

Fig 1

Quantitative immunophenotyping of peripheral lymphocyte subsets based on flow cytometric analysis of surface markers. (A) Enumeration of absolute numbers of T cells (CD3⁺, CD3⁺ CD4⁺, and CD3⁺ CD8⁺), B cells (CD20⁺) and NK cells (CD3⁻ CD16⁺ CD56⁺) per μl of blood represented as the mean ± standard error of the mean for all animals. (B) The relative distribution of each cell subset is listed as a percentage of total PBMCs. The vertical hashed line indicates the time of intravenous inoculation.

Fig 2

Longitudinal evaluation of peripheral CD4⁺ T-cell activation, proliferation, and CCR5 and integrin α4β7 surface expression. Percentages of CD4⁺ T cells expressing the early activation marker CD69 or proliferation marker Ki67 (A), late activation markers CD25 or HLA-DR (B) are shown as well as percentages of cells expressing CCR5 or coexpressing CCR5 and integrin α4β7 (C). Data points are represented as the mean ± standard error of the mean. Solid symbols and lines represent groups 1 and 2 combined, and the control group is represented with open symbols and hashed lines. Statistical significance was determined between time points for all animals combined (*, P ≤ 0.05; **, P ≤ 0.001; ***, P ≤ 0.0001). The vertical hashed line indicates the time of intravenous inoculation.

Fig 3

Longitudinal assessment of humoral immunity to VZV. VZV-specific IgG responses were evaluated by weekly VZV IgG ELISA of serum samples. Data points represent a single test per time point. The assay was conducted following stringent standard operating procedures used for clinical evaluation of anti-VZV titers in hospital patients. The ELISA readings provide a semiquantitative assessment of antibody titers. Time zero represents the initial intratracheal inoculation, and the vertical hashed line indicates the time of intravenous inoculation 12 weeks thereafter.

Fig 4

VZV-specific cell-mediated immunity. (A) IFN-γ ELISPOT assay responses in PBMC preparations following stimulation with whole VZV antigen. Data points represent the background-subtracted mean response from at least three replica wells on each of two duplicate plates per experiment with error bars indicative of the standard error of the mean. Responses are indicated as the numbers of IFN-γ-producing cells as SFC per million PBMCs. Time zero represents the initial intratracheal inoculation, and the vertical hashed line indicates the time of intravenous inoculation 12 weeks later. (B) Cytokine profile of T-cell immunity following stimulation with peptide pools spanning the VZV IE63, ORF4, gI, and gE proteins as measured by intracellular cytokine secretion of IFN-γ, MIP-1β, TNF-α, IL-2, and CD107a. Boolean gating on FlowJo software was used to determine the extent of coincident cytokine secretion. Pie charts generated using SPICE software represent the mean multifunctional responses directed against all peptide pools for each group.