On interactions between alphaherpesviruses and natural killer cells

Natural killer (NK) cells are a critical component of the innate immune response to viral infection. NK cells are responsible for the early control of virus spread, cytolytically killing infected cells, as well as secreting proinflammatory cytokines to enhance immune responses. In patients with deficiencies in NK cell function, there is an extreme susceptibility to infection with herpesviruses, in particular, varicella zoster virus (VZV) and herpes simplex virus type 1 (HSV-1). These two medically important human alphaherpesviruses cause widespread disease in human hosts, with VZV being the causative agent of varicella (chickenpox) and herpes zoster (shingles), while HSV-1 causes recurrent orolabial lesions (cold sores). Both viruses have the potential to cause severe complications, such as encephalitis and debilitating nerve pain. The vital role that NK cells play in controlling VZV and HSV-1 infections denotes an intricate struggle for dominance between virus and NK cell antiviral immunity; however, research in this area has remained surprisingly limited. This thesis explored the interactions between human NK cells and alphaherpesviruses, examining NK cell recognition of infected cells, as well as investigating viral infection of NK cells and manipulation of their function. Investigation into alphaherpesvirus interactions with NK cells first focused on examining whether VZV and HSV-1 modulated the surface of infected cells to potentially regulate NK cell recognition. In vitro co-culture of NK cells with VZV infected cells revealed that NK cells did not display enhanced activation in response to the infection, suggesting that specific viral mechanisms to limit NK cell detection may be at play. Delving into this, the expression of four specific ligands (MICA, ULBP1–3) recognised by the activating NK cell receptor, NKG2D, were examined during viral infection. Comparing VZV and HSV-1, differential patterns of regulation were found between the two viruses, as well as between distinct NKG2D ligands. Given that VZV appeared to be evading NK cell recognition, the research focus then turned to investigating how VZV directly interacted with NK cells. VZV is established as a lymphotropic virus, using the infection of immune cells to disseminate virus around the body, however it has so far remained unknown whether NK cells are permissive to VZV infection. Examination of human peripheral blood NK cells revealed that VZV productively infected NK cells, facilitating transmission of infectious virus to other cells in culture. VZV preferentially infected mature NK cell populations, as well as modulating cell-surface expression of maturityassociated markers. Notably, VZV infection of NK cells led to upregulated expression of chemokine receptors implicated in trafficking to the skin, suggesting that NK cells may play a key role in VZV pathogenesis. As NK cells were permissive to productive VZV infection, the effect of VZV on NK cell function was then investigated. Assessing cytolytic function, it was found that co-culture with VZV lead to potent inhibition of NK cell responsiveness to target cell stimulation. Remarkably, not only were VZV infected NK cells impaired, but also NK cells exposed to virus were inhibited without needing to progress to full productive infection. HSV-1 had a similar capacity to paralyse NK cell cytolytic function, identifying a powerful immune evasion strategy shared by both alphaherpesviruses. In contrast, when NK cell cytokine responses were investigated, differential targeting of cytokine production was demonstrated between VZV and HSV-1. Overall, this thesis illuminates the complex interactions that occur between viral infection and the immune response. The findings presented in this thesis enhance our understanding of how viruses like VZV and HSV-1 are able to evade the immune system to establish lifelong infections, as well as furthering our understanding of how viruses can shape and manipulate the immune response

Functional paralysis of human natural killer cells by alphaherpesviruses

Natural killer (NK) cells are implicated as important anti-viral immune effectors in varicella zoster virus (VZV) infection. VZV can productively infect human NK cells, yet it is unknown how, or if, VZV can directly affect NK cell function. Here we demonstrate that VZV potently impairs the ability of NK cells to respond to target cell stimulation in vitro, leading to a loss of both cytotoxic and cytokine responses. Remarkably, not only were VZV infected NK cells affected, but VZV antigen negative NK cells that were exposed to virus in culture were also inhibited. This powerful impairment of function was dependent on direct contact between NK cells and VZV infected inoculum cells. Profiling of the NK cell surface receptor phenotype by multiparameter flow cytometry revealed that functional receptor expression is predominantly stable. Furthermore, inhibited NK cells were still capable of releasing cytotoxic granules when the stimulation signal bypassed receptor/ligand interactions and early signalling, suggesting that VZV paralyses NK cells from responding. Phosflow examination of key components in the degranulation signalling cascade also demonstrated perturbation following culture with VZV. In addition to inhibiting degranulation, IFN-γ and TNF production were also repressed by VZV co-culture, which was most strongly regulated in VZV infected NK cells. Interestingly, the closely related virus, herpes simplex virus type 1 (HSV-1), was also capable of efficiently infecting NK cells in a cell-associated manner, and demonstrated a similar capacity to render NK cells unresponsive to target cell stimulation–however HSV-1 differentially targeted cytokine production compared to VZV. Our findings progress a growing understanding of pathogen inhibition of NK cell function, and reveal a previously unreported strategy for VZV to manipulate the immune response.This work was funded by NHMRC project grant APP1088005 awarded to AA, BS and BM. and NHMRC project grant APP1126599 awarded to DT and AA. DT was funded by NHMRC fellowship APP110432

Varicella zoster virus productively infects human natural killer cells and manipulates phenotype.

Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer (NK) cells are part of the early immune response to infection, and have been implicated in controlling VZV infection in patients. Understanding of how VZV directly interacts with NK cells, however, has not been investigated in detail. In this study, we provide the first evidence that VZV is capable of infecting human NK cells from peripheral blood in vitro. VZV infection of NK cells is productive, supporting the full kinetic cascade of viral gene expression and producing new infectious virus which was transmitted to epithelial cells in culture. We determined by flow cytometry that NK cell infection with VZV was not only preferential for the mature CD56dim NK cell subset, but also drove acquisition of the terminally-differentiated maturity marker CD57. Interpretation of high dimensional flow cytometry data with tSNE analysis revealed that culture of NK cells with VZV also induced a potent loss of expression of the low-affinity IgG Fc receptor CD16 on the cell surface. Notably, VZV infection of NK cells upregulated surface expression of chemokine receptors associated with trafficking to the skin -a crucial site in VZV disease where highly infectious lesions develop. We demonstrate that VZV actively manipulates the NK cell phenotype through productive infection, and propose a potential role for NK cells in VZV pathogenesis

Functional paralysis of human natural killer cells by alphaherpesviruses.

Natural killer (NK) cells are implicated as important anti-viral immune effectors in varicella zoster virus (VZV) infection. VZV can productively infect human NK cells, yet it is unknown how, or if, VZV can directly affect NK cell function. Here we demonstrate that VZV potently impairs the ability of NK cells to respond to target cell stimulation in vitro, leading to a loss of both cytotoxic and cytokine responses. Remarkably, not only were VZV infected NK cells affected, but VZV antigen negative NK cells that were exposed to virus in culture were also inhibited. This powerful impairment of function was dependent on direct contact between NK cells and VZV infected inoculum cells. Profiling of the NK cell surface receptor phenotype by multiparameter flow cytometry revealed that functional receptor expression is predominantly stable. Furthermore, inhibited NK cells were still capable of releasing cytotoxic granules when the stimulation signal bypassed receptor/ligand interactions and early signalling, suggesting that VZV paralyses NK cells from responding. Phosflow examination of key components in the degranulation signalling cascade also demonstrated perturbation following culture with VZV. In addition to inhibiting degranulation, IFN-γ and TNF production were also repressed by VZV co-culture, which was most strongly regulated in VZV infected NK cells. Interestingly, the closely related virus, herpes simplex virus type 1 (HSV-1), was also capable of efficiently infecting NK cells in a cell-associated manner, and demonstrated a similar capacity to render NK cells unresponsive to target cell stimulation-however HSV-1 differentially targeted cytokine production compared to VZV. Our findings progress a growing understanding of pathogen inhibition of NK cell function, and reveal a previously unreported strategy for VZV to manipulate the immune response

VZV infects both CD57^– and CD57^bright NK cells and drives CD57 expression.

<p>CD3^–CD56⁺CD57^– NK cells and CD3^–CD56⁺CD57^bright NK cells were isolated from healthy human donor PBMCs by FACS sorting and subsequently mock or VZV infected with or without IL-2 (200 U/ml) for 2 days before analysis by flow cytometry. (A) Diagram describes experimental design of isolating NK cells on CD57 expression, then infecting, and subsequently analysing for infection and phenotype changes. (B) Plots show surface VZV gE:gI expression between subsets from one representative donor. Graph shows frequency of VZV⁺ NK cell subsets when untreated or with IL-2 (shaded) for three donors. Bars indicate mean. (C) Plots show subsequent CD57 expression between mock, bystander and VZV⁺ CD57^– NK cells (left panels) and CD57 versus gE:gI expression for VZV cultured CD57^– NK cells (middle panels), from one representative donor. Graphs show frequency of CD57 expression on mock, bystander and VZV⁺ CD57^– NK cells for three donors. Bars indicate mean. *p < 0.05 (two-tailed paired t test). (D) Histograms show CD16 expression for mock, bystander and VZV⁺ CD57^– NK cells (left panel) and CD57^bright NK cells (right panel) for one representative donor (n = 3).</p

NK cells are productively infected by VZV and support virus transmission.

<p>NK cells (CD3^–CD56⁺) were FACS sorted from healthy human donor CD56⁺-selected lymphocytes following mock or VZV infection for 1 day. (A & B) Staining by IFA of sorted VZV cultured (left panels) or mock cultured (right panels) NK cells for IE63 (A), pORF29 (B) or respective isotype control, with DAPI (n = 3). (C) Sorted VZV cultured NK cells were added to ARPE-19 epithelial cell monolayers. Four days later monolayers were fixed and infectious centres detected with IFA by staining for IE63 and gE:gI, with DAPI. One representative experiment of five is shown.</p

VZV infects human peripheral blood NK cells, CD3⁺CD56⁺ lymphocytes, and T cells.

<p>Healthy human donor PBMCs were inoculated with mock or VZV infected ARPE-19 epithelial cells for 2 days then analysed for infection by flow cytometry. (A) Representative flow cytometry plots of mock or VZV-S infection, examining surface VZV gE:gI expression on live T cells (CD3⁺CD56^–), CD3⁺CD56⁺ lymphocytes, and NK cells (CD3^–CD56⁺). (B) Frequencies of live gE:gI⁺ lymphocytes in total (shaded), compared to specific populations: T cells, CD3⁺CD56⁺ lymphocytes, and NK cells (n = 19). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean. Statistical analysis was performed between specific lymphocyte populations. **p < 0.01, ****p < 0.0001 (RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey’s multiple comparisons test). (C) Representative flow cytometry plots of vOka infection, examining surface gE:gI expression on live T cells (CD3⁺CD56^–), CD3⁺CD56⁺ lymphocytes, and NK cells (CD3^–CD56⁺) (n = 3).</p

IL-2 stimulation of NK cells, CD3⁺CD56⁺ lymphocytes, and T cells enhances VZV infection.

<p>(A) Healthy human donor PBMCs were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface VZV gE:gI expression from one representative donor and graphs show frequency of live gE:gI⁺ NK cells (CD3^–CD56⁺) (top panels), CD3⁺CD56⁺ lymphocytes (middle panels), and T cells (CD3⁺CD56^–) (bottom panels). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean (n = 8). ***p < 0.001, ****p < 0.0001 (two-tailed paired t test). (B & C) Healthy human donor CD56⁺-selected lymphocytes were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface gE:gI expression from one representative donor and graphs show frequency of live gE:gI⁺ NK cells (B) or CD3⁺CD56⁺ lymphocytes (C). Symbols represent individual donors, consistent across (B & C) (n = 7). *p < 0.05 (two-tailed Wilcoxon matched-pairs signed rank test).</p

NK cell markers associated with maturity influence VZV infection of NK cells and are modulated by VZV.

<p>Healthy human donor PBMCs were mock or VZV infected with or without IL-2 (200 U/ml) for 2 days then analysed by flow cytometry. (A) Diagram describes gating strategy and tSNE analysis workflow for samples shown in (B & C). (B & C) tSNE plots show marker expression levels for single parameters on individual cells in the tSNE map for mock and VZV cultured NK cells after 2 days, either untreated (B) or in the presence of IL-2 (C). Arrowheads indicate the CD56^bright NK cell subset, and the outlined population indicates the localisation of VZV⁺ NK cells. One representative experiment of three is shown. (D & E) Plots show CD57 expression between mock and VZV cultured NK cells (D) and between bystander and VZV⁺ NK cells (E), from one representative donor. Graphs show respective frequencies of CD57⁺ NK cells when untreated or with IL-2 (shaded) for four donors. Bars indicate mean. (F) Histograms show CD16 expression for mock, bystander and VZV⁺ NK cells from one representative donor. Graph shows frequency of CD16⁺ NK cells when untreated or with IL-2 (shaded) for six donors. Bars indicate mean. *p < 0.05, **p < 0.01, ***p < 0.001 (Friedman test with Dunn’s multiple comparisons test).</p

Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency

Autosomal recessive IRF7 deficiency was previously reported in three patients with single critical influenza or COVID-19 pneumonia episodes. The patients' fibroblasts and plasmacytoid dendritic cells produced no detectable type I and III IFNs, except IFN-beta. Having discovered four new patients, we describe the genetic, immunological, and clinical features of seven IRF7-deficient patients from six families and five ancestries. Five were homozygous and two were compound heterozygous for IRF7 variants. Patients typically had one episode of pulmonary viral disease. Age at onset was surprisingly broad, from 6 mo to 50 yr (mean age 29 yr). The respiratory viruses implicated included SARS-CoV-2, influenza virus, respiratory syncytial virus, and adenovirus. Serological analyses indicated previous infections with many common viruses. Cellular analyses revealed strong antiviral immunity and expanded populations of influenza- and SARS-CoV-2-specific memory CD4(+) and CD8(+)T cells. IRF7-deficient individuals are prone to viral infections of the respiratory tract but are otherwise healthy, potentially due to residual IFN-beta and compensatory adaptive immunity