Correlation between IL-18 secretion by peripheral blood mononuclear cells and NK cells activation after SARS-CoV-2 booster vaccination in HIV-1-infected individuals

To the Editor: Natural killer (NK) cells are vital in early antiviral and antitumor immune responses and the their dysfunction is closely associated with disease severity and progression.[1] Human immunodeficiency virus-1 (HIV-1) infection leads to reduced NK cell counts, disrupted homeostasis, altered receptor expression profiles, and impaired effector functions[2]. Therefore, understanding NK cells activation and functions following SARS-CoV-2 vaccination holds significant clinical implications for developing tailored immunization and therapeutic strategies in HIV-1-infected individuals. Previous data have demonstrated potent antiviral activity of NK cells against SARS-CoV-2, mediated through direct cytotoxicity or indirect mechanisms involving cytokine secretion.[3] Innate cytokines induced by live-attenuated or inactivated vaccines and adjuvants—such as IL-12, IL-18, and type I interferon, can effectively activate NK cells and promote their differentiation into cytokine-induced memory-like NK cells.[4] Our previous research revealed that NK cell responses could be induced and peaked at week 2 post-vaccination after the booster immunization with inactivated SARS-CoV-2 vaccines.[5] However, the dynamic changes of IL-12, IL-15, IL-18 following booster vaccination and their potential involvement in NK cell activation remain poorly understood. This study was approved by Beijing Youan Hospital Research Ethics Committee (Nos. 2021-031 and 2021-079) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each subject. We enrolled 20 HIV-1-infected individuals (aged 36.50 years, CD4+ T-cell count >350 cells/μL) and 20 matched healthy controls from Beijing Youan Hospital between October 2021 and December 2021. The characteristic of these enrolled subjects was shown in Table S1, https://links.lww.com/IDI/A67. All participants had received primary and booster vaccinations with inactivated SARS-CoV-2 vaccines (Sinovac). Differences with P values less than 0.05 were considered statistically significant. As shown in Figure 1A–C, the levels of IL-12 and IL-18 secreted into supernatant were significantly upregulated after peripheral blood mononuclear cells (PBMCs) were pulsed by wild-type Spike protein compared to unstimulated cells in healthy donors (P = 0.012; P = 0.035, respectively), while IL-15 levels remained unchanged (Figure 1B). However, HIV-1-infected individuals exhibited no significant cytokine response to Spike protein stimulation (Figure 1A–C). Notably, as shown in Figure 1D–F, with Spike peptides stimulation, HIV-1-infected individuals showed elevated IL-12 (P = 0.024) and IL-15 (P = 0.023) in supernatant in relative to healthy controls before the booster injections, while no difference in IL-18. The increase of IL-12 released by PBMCs was observed in healthy controls (P = 0.023) but not in HIV-1-infected individuals in week 2 after the booster injection. To further explore the role of IL-12, IL-15 and IL-18 in NK cell responses, we analyzed correlations between the level of these cytokines secreted into supernatant by PMBCs stimulated with Spike protein and the percentage of activated and functional NK cells in week 2. Specifically, the levels of IL-18 correlated positively with the frequencies of CD69+ NK cells (r = 0.697, P = 0.005) and CD107a+ NK cells (r = 0.589, P = 0.023) in healthy controls (Figure 1G&1I). In HIV-1-infected individuals, the concentration of IL-18 retained a positive association with CD69+ NK cells (r = 0.480, P = 0.032) and CD25+ NK cells (r = 0.461, P = 0.047) but failed to correlate with CD107a expression (P = 0.660) (Figure 1G–I), suggesting a vital role of IL-18 in NK cell responses induced by SARS-CoV-2 vaccines.Figure 1: IL-18 was up-regulated after the SARS-CoV-2 Spike stimulation and was correlated with NK cell responses. (A–C) Concentrations of IL-12, IL-15, and IL-18 before and after Spike stimulation in HC (n = 10) and HIV-1-infected individuals (n = 10) at week 2 following booster vaccination with SARS-CoV-2 vaccines. Statistical comparisons were performed using paired t-tests. (D–F) Concentrations of IL-12, IL-15, and IL-18 after Spike stimulation in HC and HIV-1-infected individuals at week 0 (HC: n = 10; HIV: n = 10) and week 2 (HC: n = 17; HIV: n = 20) after booster immunization. Normally distributed data were analyzed using t-tests and are presented as means and standard deviation (D); non-normally distributed data were analyzed using Mann–Whitney U tests and are presented as medians and interquartile range (E&F). Data normality were assessed via the Shapiro-Wilk test in this study. (G–I) Correlations between the concentration of IL-18 and the frequencies of CD69+ NK cells (G), CD25+ NK cells (H), and CD107a+ NK cells (I) after Spike stimulation in HC (n = 15) and HIV-1-infected individuals (n = 19) at week 2 post-booster vaccination. Correlations were analyzed using Spearman's rank correlation test. unsti: unstimulated; sti: stimulated; IL: Interleukin; HIV: Human immunodeficiency virus; NK: Natural killer; HC: Healthy controls.In this study, we observed that Spike protein significantly upregulated IL-18 secretion by PBMCs from healthy controls, while HIV-1-infected individuals exhibited a markedly attenuated response to Spike protein stimulation. Furthermore, IL-18 levels in healthy controls showed positive correlations with the percentages of CD69+ NK, CD25+ NK and CD107a+ NK cells, indicating that IL-18 was engaged in NK cell activation and function. The activation of NK cells, as well as their subsequent maturation, proliferation, and effector functions, relies on cytokines derived from myeloid accessory cells, such as IL-12, IL-18, and type I interferons[4]. Our study indicates that the SARS-CoV-2 wild-type Spike protein can significantly upregulate the expression of IL-12 and IL-18 in the supernatant of cultured cells from healthy subjects, suggesting that these cytokines may enhance vaccine efficacy. In addition, as reported in our previous study, weaker NK cell responses were observed in HIV-1-infected individuals after booster immunization with SARS-CoV-2 vaccines compared to healthy controls.[5] In this study, no significant changes in the levels of IL-12, IL-15, or IL-18 in the supernatant were observed before and after Spike protein stimulation in HIV-1-infected patients. Considering the correlation between IL-18 expression and the function of NK cells, we speculate that one potential mechanism by which inactivated SARS-CoV-2 vaccines enhance NK cells activation may involve upregulation of IL-18 expression, whereas HIV-1-infected individuals exhibit blunted NK cell activation responses. Previous study has demonstrated that incorporation of IL-18 as an adjuvant in vaccines can enhance antibodies production and cellular immune responses.[6] Here, we found that HIV-1-infected individuals exhibit insufficient induction of IL-18, suggesting that adding IL-18 as an adjuvant may improve vaccine efficacy for HIV-1-infected patients. The main limitations of this study lie in its small sample size and the inclusion only of HIV-1-infected individuals with CD4 counts above 350 (cells/µL), as well as the assessment of IL-12, IL-15, and IL-18 solely at baseline and two weeks post-vaccination. Future studies with larger cohorts and longer follow-up are needed to definitively clarify the relationship between these cytokine changes and vaccine-induced NK-cell activation and immune function. In conclusion, our study revealed the expression profiles of IL-12, IL-15 and IL-18 after the booster vaccination and demonstrated that upregulation of IL-18 expression positively correlated with the frequencies of activated and functional NK cells. This highlights the critical role of IL-18 in mediating NK cell responses induced by inactivated SARS-CoV-2 vaccines. Funding This study was supported by the National Natural Science Foundation of China (82272319), the National Key R&D Program of China (2023YFC2308300 and 2023YFE0116000); the Beijing Natural Science Foundation (L222068), the High-Level Public Health Specialized Talents Project of Beijing Municipal Health Commission (2022-2-018), and the Beijing Key Laboratory for HIV/AIDS Research (BZ0089). Author Contributions Xiuwen Wang, Bin Su and Hao Wu designed the study, and wrote the manuscript; Xiuwen Wang, Xiaodong Yang, Xin Zhang, and Wei Xia performed experiments assay and did data analysis; Tong Zhang, Hao Wu and Bin Su conceived the study and supervised the whole study. All authors contributed to the critical review of the article before final submission, read and approved the final manuscript. Conflicts of Interest None. Data Availability Statement The data that support the findings of this study are available from the corresponding authors upon reasonable request.