Durable Natural Killer Cell Response After Three Doses of SARS-CoV-2 Inactivated Vaccine in HIV-Infected Individuals

Introduction

In late 2019, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a global pandemic, causing significant morbidity and mortality. By April 2023, SARS-CoV-2 had infected approximately 760 million people and resulted in 7 million deaths worldwide. The virus is the causative agent of coronavirus disease 2019 (COVID-19), which can lead to severe respiratory and vascular complications, including acute respiratory distress syndrome, multiple organ failure, and death. During SARS-CoV-2 infection, natural killer (NK) cells, which play a critical role in the innate immune response, exhibit functional defects and a reduction in number, which are closely associated with disease severity and prognosis.

NK cells have traditionally been considered part of the innate immune system. However, recent evidence suggests that some NK cells exhibit characteristics of adaptive immune cells, such as memory-like properties. Vaccination or infection can induce the expansion, differentiation, and persistence of these memory or adaptive NK cells, enhancing their cytotoxicity and interferon (IFN)-γ production upon subsequent stimulation. This adaptive response is crucial for the prevention and control of viral diseases.

People living with human immunodeficiency virus (PLWH) are at increased risk of severe COVID-19 outcomes due to immune dysfunction. The cellular and humoral immune responses of PLWH to vaccines, including SARS-CoV-2 vaccines, are often diminished. However, it remains unclear whether SARS-CoV-2 vaccination can induce NK cell activation, proliferation, and enhanced degranulation and IFN-γ production in PLWH. This study aimed to evaluate the dynamic changes in NK cell frequency, phenotype, and function in PLWH and healthy controls (HCs) following three doses of the SARS-CoV-2 inactivated vaccine, as well as their responsiveness to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation.

Methods

Study Participants and Samples

The study enrolled 47 HIV-infected individuals aged 18–59 years and 30 age- and sex-matched healthy controls from Beijing Youan Hospital between April 2021 and June 2022. All participants received three doses of the SARS-CoV-2 inactivated vaccine (CoronaVac, Sinovac Life Sciences, Beijing, China). Blood samples were collected from PLWH before the first vaccination (pre-vaccination) and at 0, 2, 4, and 12 weeks after the third dose. For HCs, blood samples were collected only at 0, 2, 4, and 12 weeks after the third dose, as they had already received their first dose at enrollment. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient centrifugation and cryopreserved for further analysis.

Flow Cytometry Analysis

Flow cytometry was used to analyze the phenotype and function of NK cells. PBMCs were stained with fluorophore-labeled antibodies to assess the expression of surface markers (CD3, CD16, CD25, CD56, CD57, CD69, NKG2A, NKG2C) and intracellular markers (Ki67, IFN-γ, CD107a). The gating strategy included identifying total CD3–CD56+ NK cells and further dividing them into subsets based on CD57 expression: CD56bright, CD56dimCD57–, and CD56dimCD57+.

In Vitro Stimulation with SARS-2-OS Protein

PBMCs were stimulated with 1 µg/mL of purified recombinant SARS-CoV-2 Omicron Spike (SARS-2-OS) protein for 18 hours. The expression of NK cell activation, proliferation, and degranulation markers was evaluated before and after stimulation.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 6.0. Paired t-tests or Mann-Whitney U tests were used for functional responses, depending on data distribution. Group comparisons were made using the Kruskal-Wallis test with Dunn correction. Correlation analysis was performed using Spearman’s correlation. A p-value of <0.05 was considered statistically significant.

Results

NK Cell Responses to SARS-CoV-2 Vaccination in PLWH

The frequency of CD3–CD56+ NK cells in PLWH was slightly lower at 0 weeks post-vaccination compared to pre-vaccination but increased by 12 weeks. The proportion of CD56bright and CD56dim subsets remained stable, with CD56dimCD57+ subsets being the most abundant. The frequency of CD16+ NK cells decreased post-vaccination, reaching its lowest point at 2 weeks and then gradually returning to pre-vaccination levels. The expression of CD25, an activation marker, peaked at 2 weeks post-vaccination and then declined. Similarly, the frequency of IFN-γ+ and CD107a+ NK cells peaked at 2 weeks post-vaccination, indicating enhanced NK cell activation and degranulation.

NK Cell Responses to SARS-CoV-2 Vaccination in HCs

In HCs, the frequency of CD3–CD56+ NK cells increased slightly but not significantly at 2–12 weeks post-vaccination. The frequency of CD69+ NK cells was significantly higher at 12 weeks compared to 2 weeks, particularly in the CD56dim subset. The frequency of CD107a+ NK cells peaked at 2 weeks post-vaccination and then declined. A positive correlation was observed between the frequency of CD25+ and Ki67+ NK cells, suggesting a link between NK cell activation and proliferation.

In Vitro SARS-2-OS Protein Stimulation

In PLWH, stimulation with SARS-2-OS protein significantly decreased the frequency of CD16+ NK cells at 12 weeks post-vaccination. The frequency of Ki67+ NK cells increased at 4 weeks post-vaccination, but no significant changes were observed in CD25, CD69, CD107a, or IFN-γ expression. In HCs, SARS-2-OS stimulation significantly increased the frequency of CD25+ and Ki67+ NK cells at 4 weeks and CD107a+ NK cells at 2 and 4 weeks post-vaccination.

Comparison of NK Cell Phenotype and Function Between PLWH and HCs

The frequency of CD3–CD56+ NK cells was slightly higher in PLWH than in HCs, but the difference was not significant. The proportion of CD56dimCD57– subsets was lower in PLWH, while CD56dimCD57+ subsets were higher, with significant differences at 12 weeks. The frequency of NKG2C+ NK cells was significantly higher in PLWH. The frequency of CD16+ NK cells was significantly higher in PLWH at 2, 4, and 12 weeks post-vaccination, while the frequency of CD25+ and CD69+ NK cells was significantly lower in PLWH. No significant differences were observed in Ki67, IFN-γ, or CD107a expression between PLWH and HCs.

Discussion

This study provides a comprehensive analysis of NK cell responses to SARS-CoV-2 vaccination in PLWH and HCs. The results demonstrate that three doses of the SARS-CoV-2 inactivated vaccine elicit durable NK cell activation, proliferation, and degranulation in both groups. However, PLWH exhibited altered NK cell phenotypes and reduced responsiveness to SARS-2-OS protein stimulation compared to HCs.

The frequency of CD16+ NK cells decreased post-vaccination in both groups, likely due to NK cell activation and cytokine secretion. The frequency of CD25+ and CD69+ NK cells was lower in PLWH, indicating reduced NK cell activation in this population. The proportion of CD56dimCD57+ subsets was higher in PLWH, suggesting a shift toward more differentiated NK cells. The frequency of NKG2C+ NK cells was significantly higher in PLWH, possibly due to cytomegalovirus (CMV) co-infection.

In vitro stimulation with SARS-2-OS protein enhanced NK cell proliferation and degranulation in HCs but had limited effects in PLWH. This finding highlights the reduced responsiveness of NK cells in PLWH to activating stimuli, which may have implications for vaccine efficacy in this population.

Conclusion

Three doses of the SARS-CoV-2 inactivated vaccine induce durable NK cell responses in both PLWH and HCs. The vaccine enhances NK cell activation, proliferation, and degranulation, although PLWH exhibit altered NK cell phenotypes and reduced responsiveness to SARS-2-OS protein stimulation. These findings have important implications for the use of NK cells in immunotherapy for COVID-19-related diseases and underscore the need for further research to optimize vaccine strategies for immunocompromised populations.

doi.org/10.1097/CM9.0000000000002947

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