Specific human cytomegalovirus signature detected in NK cell metabolic changes post vaccination

Effective vaccines for human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) remain a significant challenge for these infectious diseases. Given that the innate immune response is key to controlling the scale and nature of developing adaptive immune responses, targeting natural killer (NK) cells that can promote a T-helper type 1 (Th1)-type immune response through the production of interferon-γ (IFNγ) remains an untapped strategic target for improved vaccination approaches. Here, we investigate metabolic and functional responses of NK cells to simian adenovirus prime and MVA boost vaccination in a cohort of healthy volunteers receiving a dual HCV-HIV-1 vaccine. Early and late timepoints demonstrated metabolic changes that contributed to the sustained proliferation of all NK cells. However, a strong impact of human cytomegalovirus (HCMV) on some metabolic and functional responses in NK cells was observed in HCMV seropositive participants. These changes were not restricted to molecularly defined adaptive NK cells; indeed, canonical NK cells that produced most IFNγ in response to vaccination were equally impacted in individuals with latent HCMV. In summary, NK cells undergo metabolic changes in response to vaccination, and understanding these in the context of HCMV is an important step towards rational vaccine design against a range of human viral pathogens.


Statistics
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Software and code
Policy information about availability of computer code Data collection FACS DIVA was used to collect Flow cytometry data. Data analysis Flowjo v10.6.1 and GraphPad Prism 9 were used to analyse data.
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Data
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Life sciences study design
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We have original power calculations and these vary depending on the assay. We have been working in this area for almost 20 years and use statistics appropriate to the data being presented. With human studies, our samples usually range between 5-20 for individual experimental data sets.
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Replication
We have internal replicates where appropriate e.g. ELISA. All the data presented are the independent biological replicates.

Recruitment
Recruited by collaborators in Oxford as part of Peach I clinical trial as above. Unvaccinated donors were recruited in TBSI with no selection bias other than to be HCMV positive.

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Ethics oversight
Approvals for the clinical trial study from which samples were used was as previously reported (19). Ethics for this study was provided by the REC of St. James's Hospital, Dublin 8 Tallaght Hospital / St James's Hospital Joint Research Ethics Committee (reference 2014/07/List 27) and for the healthy HCMV+ donors by the REC of School of Biochemistry and Immunology, Trinity College, Dublin 2 (reference BI-CG-311220).
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Flow Cytometry
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Methodology Sample preparation
For studies on unvaccinated HCMV+ donors human plasma was isolated by centrifugation, frozen and screened for HCMV anti-pp65 IgG. Peripheral blood mononuclear cells were isolated from blood(40ml) using Lymphoprep . Cryopreserved PBMC from the PEACHI 04 Phase I Clinical Trial (Hartnell et al, 2018 PMID: 30713538) were thawed, washed and rested for 2 hours in RPMI-1640 (GIBCO) at 37°C, 5% CO2 for ex vivo analyses or for cytokine production analysis cells were stimulated with IL12 (30ng/ml, Miltenyi Biotec)/ IL15 (100ng/ml, NCI Institute) at 37°C for 18 hours with GolgiPlug (BD Pharmingen) for the final 4 hours. Cells treated for 18 hours with cytokine plus Rapamycin (20nM) were included as a negative control for pS6 staining when cell numbers allowed.
For flow cytometry staining: Cells were stained with NIR-LIVE/ DEAD (Life Technologies) for 10 minutes then washed with PBS, 5% FBS, twice. Cells were then stained with antibodies diluted in PBS, FBS 5% for 20 minutes at 4C. Cells were then washed twice before data collection. For intracellular staining, following surface staining, cells were fixed and permeabilized (20 minutes) (BD Cytofix/cytoperm). Fixed cells were then incubated with antibodies for 30 minutes at 4C. Cells were then washed twice before data collection.

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April 2020 Cryopreserved serum or plasma was thawed on ice before ELISA analysis for cytokines IFN-y, IL-2, IL-12 and IL-15 (BIolegend), as well as HCMV anti-pp65 IgG (Alpha Diagnostics).

Instrument
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Cell population abundance
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Gating strategy
Global cell population is gated on FSC-A/SSC-A. Single cells are gated on FSC-A/FSC-W. Live cells -using NIR LIVE/DEAD are gated. The dead population is determined using cells pretreated with ethanol to induce cell death as a positive control. NK cells were gated as CD56+/CD3-. The IFNg+ and p-S6+ populations were determine using the corresponding negative population in non-stimulated cells or fluorescence minus one controls.
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Magnetic resonance imaging
Experimental design