Viral Load, Clinical Disease Severity and Cellular Immune Responses in Primary Varicella Zoster Virus Infection in Sri Lanka

BACKGROUND: In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied. METHODOLOGY: We used IFNgamma ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection. PRINCIPAL FINDINGS: Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNgamma ELISpot responses of patients (P<0.001, r = -0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation. CONCLUSIONS: Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses

Relation of age to number of lesions.

<p>Relation of age to number of lesions.</p

CD4+ T cells represent the main source of VZV-specific IFNγ production during acute infection.

<p>Example of <i>ex vivo</i> intracellular cytokine analysis of PBMC of a patient with acute varicella infection. The VZV vaccine was used as the antigenic stimulus. Cells are gated on CD3+.</p

Clinical characteristics of cohort.

<p>Clinical characteristics of cohort.</p

Relation of clinical characteristics to viral load.

<p>(A) Viral loads in patients with primary VZV who were 25 years or younger compared to older individuals. (B) Quantity of viral copies in the peripheral blood of patients with varying severity of primary VZV infection. (C) Quantity of viral copies in patients with primary VZV infection with different numbers of skin lesions. Line shows median.</p

VZV-specific CD4+ T cells phenotypic analysis.

<p>Comparison of the expression of CLA (cutaneous lymphocyte associated antigen-1), lymph node homing markers and activation markers on the tetramer-binding cells (circles) and total CD4+ population (triangles). Cells are gated on CD4+ Via probe⁻CD14⁻CD19⁻ cells.</p

Clinical and laboratory characteristics of patients with acute dengue infection.

<p>Clinical and laboratory characteristics of patients with acute dengue infection.</p

Distribution of SS responses in those who responded to at least one SS peptide of the 4 dengue virus serotype in patients with acute infection.

<p>A indicates, responses to SS peptides during acute infection and C indicated responses to SS during the convalescent period.</p

Effect of IL-10 blockade on antiviral T cell responses.

<p>A: <i>Ex vivo</i> IFNγ responses to DENV-NS3 overlapping peptides and FEC pool of peptides in patients with acute dengue infection, with and without IL-10 blockade (n = 10). The boxes represent the means and the horizontal bars the SEM. B: <i>Ex vivo</i> IFNγ responses to DENV-NS3 overlapping peptides in a patient with acute dengue viral infection, with and without IL-10 blockade. The left two wells are responses to NS3 without IL-10 blockade and the right two wells are responses to NS3 with IL-10 blockade. C: CD107a expression on CD8+ T cells in patients with acute dengue infection in the absence and the presence of IL-10 blockade (n = 6). The cells were gated on CD3+, CD8+ and Propidium Iodide negative cells. The PBMC were stimulated with DENV-NS3 overlapping peptides. The boxes represent the means and the horizontal bars the SEM. D. A dot plot of CD107a expression on CD3+ T cells in a patient with acute dengue infection. The cells were gated on CD3+ and Propidium Iodide negative cells. The PBMC were stimulated with DENV-NS3 overlapping peptide. The left dot-plot shows the responses toNS3 without IL-10 blockade and the right dot-plot is with IL-10 blockade. E: TNFα production by CD8+ T cells in patients with acute dengue infection in the absence and the presence of IL-10 blockade (n = 6). The cells were gated on CD3+ and CD8+ T cells. The PBMC were stimulated with DENV-NS3 overlapping peptides. The boxes represent the means and the horizontal bars the SEM. F: A dot plot of TNFα production in a patient with acute dengue infection. The cells were gated on CD3+ cells. The PBMC were stimulated with DENV-NS3 overlapping peptides. The left dot-plot shows the responses toNS3 without IL-10 blockade and the right dot-plot is with IL-10 blockade.</p