Durable natural killer cell response after three doses of SARS-CoV-2 inactivated vaccine in HIV-infected individuals

Abstract

Background:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine can induce a potent cellular and humoral immune response to protect against SARS-CoV-2 infection. However, it was unknown whether SARS-CoV-2 vaccination can induce effective natural killer (NK) cell response in people living with human immunodeficiency virus (PLWH) and healthy individuals.

Methods:

Forty-seven PLWH and thirty healthy controls (HCs) inoculated with SARS-CoV-2 inactivated vaccine were enrolled from Beijing Youan Hospital in this study. The effect of SARS-CoV-2 vaccine on NK cell frequency, phenotype, and function in PLWH and HCs was evaluated by flow cytometry, and the response of NK cells to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation was also evaluated.

Results:

SARS-CoV-2 vaccine inoculation elicited activation and degranulation of NK cells in PLWH, which peaked at 2 weeks and then decreased to a minimum at 12 weeks after the third dose of vaccine. However, in vitro stimulation of the corresponding peripheral blood monocular cells from PLWH with SARS-2-OS protein did not upregulate the expression of the aforementioned markers. Additionally, the frequencies of NK cells expressing the activation markers CD25 and CD69 in PLWH were significantly lower than those in HCs at 0, 4 and 12 weeks, but the percentage of CD16⁺ NK cells in PLWH was significantly higher than that in HCs at 2, 4 and 12 weeks after the third dose of vaccine. Interestingly, the frequency of CD16⁺ NK cells was significantly negatively correlated with the proportion of CD107a⁺ NK cells in PLWH at each time point after the third dose. Similarly, this phenomenon was also observed in HCs at 0, 2, and 4 weeks after the third dose. Finally, regardless of whether NK cells were stimulated with SARS-2-OS or not, we did not observe any differences in the expression of NK cell degranulation markers between PLWH and HCs.

Conclusion:

s:SARS-CoV-2 vaccine elicited activation and degranulation of NK cells, indicating that the inoculation of SARS-CoV-2 vaccine enhances NK cell immune response.

Introduction

In the late 2019, a new zoonosis pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged and spread rapidly around the world.^[1] By the end of April 2023, it has caused about 760 million infections and 7 million deaths, which is still one of the most serious public health challenges in the world.^[2] SARS-CoV-2 is the causative agent of coronavirus disease 2019 (COVID-19), a respiratory and vascular system disease, in severe cases, can lead to acute respiratory distress syndrome, multiple organ failure, and death. During SARS-CoV-2 infection, the number of natural killer (NK) cells decreases, functional defects occur, and are closely related to disease severity and prognosis.

NK cells have historically been thought to be part of the innate immune system; however, over the past decade, there has been growing evidence that at least some NK cells exhibit certain characteristics of adaptive immune cells.^[3,4] Vaccination or infection-elicited inflammatory cytokines and viral products promote the expansion, differentiation, and persistence of memory or adaptive NK cells, thereby enhancing NK cytotoxicity and interferon (IFN)-γ production in subsequent stimulation, which is of great significance for the prevention and control of viral diseases.^[5,6]

Additionally, people living with human immunodeficiency virus (PLWH) have an increased risk of severity, hospitalization, and mortality after SARS-CoV-2 infection due to immune deficiency, and the cellular and humoral immune responses of PLWH to various vaccines, including SARS-CoV-2 vaccine, are dramatically reduced. It is unclear whether SARS-CoV-2 vaccine can induce NK cell activation and proliferation, as well as enhanced degranulation and IFN-γ production of NK cells in PLWH. To better understand the effect of SARS-CoV-2 vaccination on NK cell immune responses in PLWH, we evaluated the dynamic changes in the frequency, phenotype, and function of NK cells, and their responsiveness to SARS-CoV-2 Omicron Spike (SARS-2-OS) protein stimulation pre-vaccination and 0–12 weeks after the third dose of SARS-CoV-2 vaccine.

Methods

Ethical statement

The study was approved by Beijing Youan Hospital Research Ethics Committee (Nos. 2021-031 and 2021-079), and written informed consent was obtained from each subject. The methods used conformed to approved guidelines and regulations.

Study participants and samples

Forty-seven human immunodeficiency virus (HIV)-infected individuals aged between 18 and 59 years who voluntarily received SARS-CoV-2 inactivated vaccine (CoronaVac, Sinovac Life Sciences, Beijing, China.) were enrolled from the Clinical and Research Center for Infectious Diseases of Beijing Youan Hospital from April 2021 to June 2022. At the same time, 30 healthy individuals who received the same vaccine regimen matched by sex and age were enrolled from the health worker of Beijing Youan Hospital. The clinical data of the subjects were collected, including: gender, age, time of confirmed HIV infection, recent CD4⁺ T cell count, HIV viral load, antiretroviral therapy (ART) regimen and duration, and opportunistic infections (such as cytomegalovirus, Epstein–Barr virus, cryptococcus neoformans, toxoplasma gondii, and mycobacterium tuberculosis). We excluded patients with major organ dysfunction, organ transplant recipients, with malignant tumors or undergoing chemotherapy, or who use immunosuppressants.

Blood samples of 47 PLWH were obtained before first vaccination (pre), and 0, 2, 4, and 12 weeks after the third dose of vaccination. Since healthy controls (HCs) had already received the first dose of SARS-CoV-2 vaccine at the time of enrollment, we only collected peripheral blood samples of them at 0–12 weeks after receiving the third dose of vaccine. Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Hypaque (GE Healthcare Life Sciences, Pittsburgh, PA, USA) density gradient centrifugation and cryopreserved in liquid nitrogen until use.

CD4⁺ T cell count and viral load measurements

Peripheral lymphocyte counts (cells/mL) were measured by 4-color flow cytometry using the human CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC reagent (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The plasma HIV-1 viral load (copies/mL of plasma) was quantified by real-time polymerase chain reaction (Abbott, Des Plaines, IL, USA). The cutoff value of this assay was 40 copies/mL.

Cell culture and flow cytometry

Cryopreserved PBMCs were thawed, washed in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 100 U/mL penicillin/streptomycin, 10% fetal bovine serum (FBS) and 20 mmol/L L-glutamine (Gibco; ThermoFisher, Waltham, MA, USA), and rested for 5 h. For cell culture, 1 × 10⁶ PBMCs from each participant were stimulated with 1 μg/mL purified recombinant SARS-CoV-2 B.1.1.529 (Omicron) spike (SARS-2-OS) receptor binding domain protein, which was prepared in Hek293F cells (Sino Biological, Beijing, China), or with RPMI 1640 medium as negative control for 18 h at 37°C, 5% CO₂. Simultaneously, anti-CD107a-PE/Cy7 (clone H4A3; Biolegend, San Diego, CA, USA) was added in the entire culture period, while GolgiStop (Monensin; 1:1500 concentration; BD Biosciences) and GolgiPlug (Brefeldin A; 1:1000 final concentration; BD Biosciences) were added for the final 5 h of culture. Cell culture supernatants were collected prior to adding GolgiStop and GolgiPlug.

Cells were stained in 96-well round-bottom plates, as described elsewhere.^[7] Briefly, cells were blocked with FcR Blocking Reagent (Miltenyi Biotech, Bergisch Gladbach, Germany) and stained with fluorophore-labeled antibodies for surface markers, including a viability marker (Horizon Fixable Viability Stain 510; BD Biosciences) in FACS buffer (eBioscience, ThermoFisher , Waltham, MA, USA) for 30 min in flow cytometry tubes. Cells were then washed in FACS buffer, fixed, and permeabilized using Cytofix/Cytoperm Kit (BD), and then stained for intracellular markers with further FcR blocking, washed again, resuspended in FACS buffer, acquired using a BD LSRII flow cytometer and FACSDiva software, and analyzed using FlowJo V10 (Tree Star, Ashland, OR, USA). FACS gates were set using unstimulated cells or Fluorescence Minus One (FMO) control, a minimum cutoff was determined as the frequency of responding NK cells in the presence of FCS alone, and samples with <1000 NK cell events were excluded from the analysis.

The following fluorophore-labeled antibodies were used: anti-CD3-AF700 (clone HIT3a), anti-CD16-PerCP (clone 3G8), anti-CD25-PE/Cy5 (clone BC96), anti-CD56-BV785 (clone 5.1H11), anti-CD57-PB (clone HNK1), anti-CD69-BV605 (clone FN50), anti-CD159c (NKG2C)-PE (clone S19005E), anti-Ki-67-BV711 (clone Ki-67), anti-IFN-γ-APC/Cy7 (clone 4S.B3) (all Biolegend), and anti-CD159a (NKG2A)-FITC (clone REAL283) (Miltenyi Biotech, Bergisch Gladbach, Germany).

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad, San Diego, CA, USA). Functional responses were compared using paired t test if the data follow a normal distribution, otherwise the Mann–Whitney U test is used. The Kruskal–Wallis test with Dunn correction was used for comparisons between groups. For correlation analysis, a linear regression model was fitted in prism and r and P values were determined using Spearman’s correlation analysis. All statistical tests were two sided. Significance levels were considered statistically significant when the P value was <0.05 in two-tailed test.

Results

Demographic and clinical characteristics of the participants

Forty-seven HIV-infected individuals aged 18–59 years and 30 gender- and age-matched healthy subjects were enrolled in this study from April 2021 to June 2022. The number of NK cells among these subjects were all >1000 events when acquiring data by flow cytometry. The median time of diagnosing HIV infection in PLWH was 6.2 years (interquartile range [IQR], 2.5–10.2), the median CD4⁺ T cell count was 597.5 (IQR, 483.2–814.7) cells/μL, and the CD4⁺ T cell/CD8⁺ T cell ratio was 0.74 (IQR, 0.61–1.15). All PLWH received effective ART and achieved complete virologic suppression for at least one year. None of the participants were infected with SARS-CoV-2 before or 12 weeks after the third dose of vaccine, and the baseline characteristics of the two groups were comparable [Table 1].

Table 1.

Baseline characteristics of enrolled PLWH and HCs who have received three doses of SARS-CoV-2 inactivated vaccine.

Characteristics	PLWH (n = 47)	HCs (n = 30)	Statistical values	P values
Male/female	43/4	25/5	1.086*	0.17
Age (years)	34.7 (31.0–38.2)	38.5 (35.0–48.5)	–1.842†	0.36
Time of diagnose: HIV infection (years)	6.2 (2.5–10.2)	NA	NA	NA
CD4+ T cell counts (cells/μL)	597.5 (483.2–814.7)	NA	NA	NA
CD4+ T cell /CD8+ T cell ratio	0.74 (0.61–1.15)	NA	NA	NA
ART regimen
2 NRTI + NNRTI	32 (68%)	NA	NA	NA
2 NRTI + INSTI	11 (23%)	NA	NA	NA
2 NRTI + PIs	3 (6%)	NA	NA	NA
NRTI + NNRTI	1 (2%)	NA	NA	NA
Laboratory examination
White blood cell count (×109/L)	6.14 (5.23–6.74)	6.92 (6.05–7.91)	–2.057†	0.41
Lymphocyte count (×109/L)	2.08 (1.76–2.56)	2.07 (1.82–2.26)	0.009†	0.11
Platelet count (×109/L)	242.00 (209.25–283.50)	249.51 (200.71–310.50)	–0.036†	0.21
ALT (U/L)	25.00 (17.00–37.75)	24.00 (17.75–46.50)	–0.295†	0.27
AST (U/L)	26.50 (21.25–32.00)	24.50 (20.00–35.75)	0.121†	0.11

Data were presented as n (%) or median (IQR). ^*Z values; ^†t values. ALT: Alanine aminotransferase; ART: Antiretroviral therapy; AST: Aspartate aminotransferase; HCs: Healthy controls; HIV: Human immunodeficiency virus; INSTI: Integrase chain transfer inhibitors; IQR: Interquartile range; NA: Not applicable; NNRTI: Non-nucleoside reverse transcriptase inhibitors; NRTI: Nucleoside reverse transcriptase inhibitors; PIs: Protease inhibitor; PLWH: people live with human Immunodeficiency virus.

SARS-CoV-2 vaccine induced robust NK cell responses in both groups

Ex vivo flow cytometric analysis was performed on CD3^–CD56⁺ NK cells from PBMCs collected pre-vaccination (only PLWH), and 0, 2, 4, and 12 weeks after the third dose. According to the expression of CD3 and CD56, total CD3^–CD56⁺ NK cells were gated first, and then NK cells were further divided into three subsets according to the expression of CD57: CD56^bright, CD56^dimCD57^–, and CD56^dimCD57⁺ subsets (CD56^bright represents the least differentiated subsets, and CD56^dimCD57⁺ represents the most differentiated subsets).^[7] The expression of Ki67 (a marker of proliferation), Fcγ receptor III (CD16), CD25 (a component of the IL-2R complex and marker of late activation), CD69 (a marker of early activation), and NK cell inhibitory receptor and activation receptors NKG2A and NKG2C were analyzed for each subset, and the flow cytometry gating strategy is shown in Figure 1.

Figure 1.

Flow cytometry gating strategy for in vitro NK cell phenotype analysis. The gating strategy for the total number of NK cells, CD56^bright, CD56^dimCD57^–, and CD56^dimCD57⁺ NK cell subsets in a representative subject. The expressions of CD16, CD25, CD69, Ki67, NKG2A, NKG2C, CD107a, and IFN-γ were analyzed for each participant. FSC-A: forward scatter angle; IFN: Interferon; NK: Natural killer; SSC-A: Side scatter angle.

We found that the frequency of CD3^–CD56⁺ NK cells in PLWH at 0 week of the third dose of vaccine was small but significantly lower than that at pre-vaccination and 12 weeks after the third dose [Figure 2A]. There was no significant change in the proportion of CD56^bright and CD56^dim (including CD56^dimCD57^– and CD56^dimCD57⁺) subsets before and after vaccination [Figure 2B, C]. In addition, the lower the differentiation of NK cells, the lower the proportion of CD3^–CD56⁺ NK cells (CD56^bright <CD56^dimCD57^– <CD56^dimCD57⁺) [Figure 2B, C]. The frequency of CD16⁺ NK cells in PLWH gradually decreases after vaccination, reaching its lowest point at 2 weeks after the third dose, and then gradually returns to the pre-vaccination levels [Figure 2D]. SARS-CoV-2 vaccination has no effect on CD16 mean fluorescence intensity (MFI) in CD3^–CD56⁺ NK cells (data not shown). Basal CD16 expression on NK cell subsets increased with increasing differentiation status (CD56^bright <CD56^dimCD57^– <CD56^dimCD57⁺), which was also consistent with the most highly differentiated CD56^dimCD57⁺ subsets having highest cytotoxicity [Figure 2E, F]. The frequency of CD25⁺ NK cells gradually increases after vaccination, peaks at 2 weeks after the third dose, and then gradually decreases to pre-vaccination levels [Figure 2G]. Conversely, the proportion of CD25-expressing NK cell subsets gradually decreases as NK cells differentiate (CD56^brigh >CD56^dimCD57^– >CD56^dimCD57⁺) [Figure 2H, I]. The frequency of IFN-γ⁺ NK cells gradually increase after vaccination, reaching a peak point at 2 weeks after the third dose, which was significantly higher than the proportion of IFN-γ⁺ NK cells 12 weeks after the third dose [Figure 2J]. Similarly, the proportion of CD107a⁺ NK cells in PLWH gradually increased after vaccination, peaked at 2 weeks after the third dose, and significantly higher than that at pre-vaccination and 12 weeks after the third dose [Figure 2K]. In addition, the proportion of CD25⁺ NK cells were positively correlated with the frequency of Ki67⁺ NK cells at 4 weeks and 12 weeks after the third dose, further suggesting an association between NK cell activation and proliferation in response to vaccination [Figure 2L, M]. No effect of SARS-CoV-2 vaccine on the frequency of NK cells expressing Ki67, CD69, NKG2A, and NKG2C, as well as the CD107a MFI of NK cells in PLWH was observed (data not shown).

Figure 2.

NK cell responses to SARS-CoV-2 vaccination in HIV-infected individuals. Flow cytometry was used to analyze the phenotype and function of NK cells in HIV-infected individuals before vaccination, at 0 week, 2 weeks, 4 weeks, and 12 weeks after the third dose of SARS-CoV-2 vaccine (n = 47). (A) the frequency of CD3^–CD56⁺ NK cells; (B) the frequency of CD56^bright and CD56^dim subset; (C) the frequency of CD56^dimCD57^– and CD56^dimCD57⁺ subset; (D) the frequency of CD16⁺ NK cells; (E) the frequency of CD56^brightCD16⁺ and CD56^dimCD16⁺ subset; (F) the frequency of CD56^dimCD57^–CD16⁺ and CD56^dimCD57⁺CD16⁺ subset; (G) the frequency of CD25⁺ NK cells; (H) the frequency of CD56^brightCD25⁺ and CD56^dimCD25⁺ subset; (I) the frequency of CD56^dimCD57^-CD25⁺ and CD56^dimCD57⁺CD25⁺ subset; (J) the frequency of IFN-γ⁺ NK cells; (K) the frequency of CD107a⁺ NK cells; (L, M) the correlation between the frequency of CD25⁺ NK cells and the proportion of Ki67⁺ NK cells at 4 weeks (L) and 12 weeks (M) after the third dose of vaccine. ^*P <0.05, ^†P <0.01, ^‡P <0.001. IFN: Interferon; NK: Natural killer; Pre: Before first vaccination; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

In healthy subjects, the frequency of CD3^–CD56⁺ NK cells was gradually increased at 2–12 weeks after the third dose of vaccine compared to that at day 0 of the third dose, but the difference was not statistically significant [Figure 3A–C]. The frequency of CD69⁺ NK cells at 12 weeks after the third dose was significantly higher than that at 2 weeks after the third dose, especially in the CD56^dim subset [Figure 3D–F]. However, the proportion of less-differentiated (CD56^bright) NK cells expressing CD69 was not changed [Figure 3E–F]. The frequency of CD107a⁺ NK cells was gradually increased after SARS-CoV-2 vaccination, and peaked at 2 weeks after the third dose, which was significantly higher than that at 12 weeks after the third dose [Figure 3G]. In addition, we observed a significant positive correlation between the percentage of CD25⁺ NK cells and the frequency of Ki67⁺ NK cells at 0, 4 , and 12 weeks after the third dose [Figure 3H–J]. Similarly, there was no significant effect of SARS-CoV-2 vaccination on the frequency of NK cells expressing Ki67, CD16, CD25, IFN-γ, NKG2A, and NKG2C in HCs (data not shown).

Figure 3.

NK cell responses to SARS-CoV-2 vaccination in healthy subjects. Flow cytometry was used to analyze the phenotype and function of NK cells in HCs at 0 week, 2 weeks, 4 weeks, and 12 weeks after the third dose of SARS-CoV-2 vaccine (n = 30). (A) the frequency of CD3^–CD56⁺ NK cells; (B) the frequency of CD56^bright and CD56^dim subset; (C) the frequency of CD56^dimCD57^– and CD56^dimCD57⁺ subset; (D) the frequency of CD69⁺ NK cells; (E) the frequency of CD56^brightCD69⁺ and CD56^dimCD69⁺ subset; (F) the frequency of CD56^dimCD57^–CD69⁺ and CD56^dimCD57⁺CD69⁺ subset; (G) the frequency of CD107a⁺ NK cells; (H–J) the correlation between the frequency of CD25⁺ NK cells and the frequency of Ki67⁺ NK cells at 0 week (H), 4 weeks (I); and 12 weeks (J) after the third dose of vaccine. ^*P <0.05, ^†P <0.01, ^‡P <0.001. HCs: Healthy controls; NK: Natural killer; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

In vitro SARS-2-OS protein stimulation induced NK cell activation, proliferation, and degranulation in HCs, but only proliferation in PLWH

To determine the effect of SARS-CoV-2 vaccination on NK cell responses to soluble SARS-2-OS protein, PBMCs collected at pre-vaccination and at 0–12 weeks after the third dose of vaccine were cultured for 18 h with 1 μg/mL SARS-2-OS protein.

In PLWH, stimulation with SARS-2-OS protein induced a remarkable decrease in the proportion of NK cells expressing CD16 and CD16 MFI compared with unstimulated cultures (medium alone) only at 12 weeks after the third dose [Figure 4A,B]. In addition, a significant increase in the proportion of Ki67⁺ NK cells was observed at 4 weeks after the third dose after stimulation with SARS-2-OS protein [Figure 4C]. However, SARS-2-OS protein stimulation had no effect on NK cells CD25, CD69, CD107a or IFN-γ expression in PLWH [Figure 4D–H]. There was a significant inverse correlation between the percentage of CD16⁺ NK cells and the frequency of CD107a⁺ NK cells at 0, 2, 4, and 12 weeks after the third dose of vaccine in PLWH after SARS-2-OS protein stimulation in vitro [Figures 4I–L]. The frequency of CD16⁺ NK cells was significantly negatively correlated with the proportion of CD25⁺ NK cells at 0 week and 2 weeks after the third dose [Figure 4M,N]. Conversely, we observed a positive correlation between the proportion of CD25⁺ NK cells and the frequency of Ki67⁺ NK cells in PLWH pre-vaccination and at 12 weeks after the third dose after SARS-2-OS protein in vitro stimulation [Figure 4O,P].

Figure 4.

Upregulated expression of Ki67, while downregulation expression of CD16 on NK cells in HIV-infected individuals in response to SARS-2-OS protein stimulation in vitro. PBMCs from baseline (pre), 0 week, 2 weeks, 4 weeks, and 12 weeks were stimulated with SARS-2-OS protein or left unstimulated (medium) for 18 h (n = 47). Cells were stained for NK cell phenotype and function markers and analyzed by flow cytometry. (A) The frequency of CD16⁺ NK cells, (B) the MFI of CD16⁺ NK cells, (C) the frequency of Ki67⁺ NK cells, (D) the frequency of CD25⁺ NK cells, (E) the frequency of CD69⁺NK cells, (F) the frequency of IFN-γ⁺ NK cells, (G) the frequency of CD107a⁺ NK cells; (H) and the MFI of CD107a⁺ NK cells before and after stimulated with SARS-2-OS protein, respectively. (I–L) the correlation between the percentage of CD16⁺ NK cells and the proportion of CD107a⁺ NK cells at (I) 0 weeks, (J) 2 weeks, (K) 4 weeks, and (L) 12 weeks after the third dose after SARS-2-OS protein in vitro stimulation, respectively. (M,N) The correlation between the percentage of CD16⁺ NK cells and the percentage of CD25⁺ NK cells at 0 weeks (M) and 2 weeks (N) after the third dose with SARS-2-OS protein in vitro stimulation, respectively. (O,P) The correlation between the percentage of CD25⁺ NK cells and the percentage of Ki67⁺ NK cells at baseline (pre-vaccination) (O) and 12 weeks (P) after the third dose after SARS-2-OS protein in vitro stimulation, respectively. The black hollow circle indicates that PBMCs was only cultured in the medium, while the blue solid circle indicates that PBMCs received SARS-2-OS protein stimulation for 18 h. ^*P <0.05, ^†P <0.01, ^‡P <0.001. HIV: Human immunodeficiency virus; IFN: Interferon; MFI: Mean fluorescence intensity; NK: Natural killer; PBMCs: Peripheral blood mononuclear cells; Pre: Before first vaccination; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; SARS-2-OS: SARS-CoV-2 Omicron Spike.

In HCs, SARS-2-OS protein in vitro stimulation resulted in a significant downregulation of the proportion of CD16⁺ NK cells, but the MFI of CD16⁺ NK cells was not altered [Figure 5A,B]. The frequency of CD25⁺ NK cells was significantly increased at 4 weeks after the third dose [Figure 5C], and the proportion of Ki67⁺ NK cells was significantly elevated at 4 weeks and 12 weeks after the third dose of vaccine with SARS-2-OS protein in vitro stimulation [Figure 5D]. Furthermore, a significant upregulation of the frequency of CD107a⁺ NK cells was observed at 2 weeks and 4 weeks after the third dose of vaccine [Figure 5E]. Similarly, the CD107a MFI of NK cells was also significantly increased at 2 weeks after the third dose of vaccine when the PBMCs was stimulated with SARS-2-OS protein in vitro for 18 h [Figure 5F]. However, in vitro stimulation of SARS-2-OS protein had no effect on CD69 [Figure 5G] and IFN-γ [Figure 5H] expression of NK cells in HCs. The frequency of NK cells expressing CD16 was significantly negatively correlated with the proportion of CD107a⁺ NK cells at 0, 2, and 4 weeks after the third dose of vaccine in HCs stimulated by SARS-2-OS protein [Figure 5I–K]. There was a significantly negative correlation between the frequency of CD25⁺ NK cells and the percentage of CD16⁺ NK cells [Figure 5M], and a positive correlation between with the proportion of Ki67⁺ NK cells [Figure 5N] at 4 weeks after the third dose in HCs with SARS-2-OS protein stimulation in vitro.

Figure 5.

Upregulated expression of CD25, Ki67, CD107a, and downregulation of CD16 on NK cells in healthy subjects in response to SARS-2-OS protein stimulation in vitro. PBMCs collected at 0 week, 2 weeks, 4 weeks, and 12 weeks after the third dose of vaccine were stimulated with SARS-2-OS protein or left unstimulated (medium) for 18 h (n = 30). Cells were stained for NK cell phenotype and function markers and analyzed by flow cytometry. (A) The frequency of CD16⁺ NK cells, (B) the MFI of CD16⁺ NK cells, (C) the frequency of CD25⁺ NK cells, (D) the frequency of Ki67⁺ NK cells, (E) the frequency of CD107a⁺ NK cells, (F) the MFI of CD107a⁺ NK cells, (G) the frequency of CD69⁺ NK cells, (H) and the frequency of IFN-γ⁺ NK cells, respectively. (I-L) The correlation between the frequency of CD16⁺ NK cells and the frequency of CD107a⁺ NK cells at 0 weeks (I), 2 weeks (J), 4 weeks (K) and 12 weeks (L) after the third dose after SARS-2-OS protein in vitro stimulation, respectively. (M) The correlation between the frequency of CD16⁺ NK cells and the frequency of CD25⁺ NK cells at 4 weeks after the third dose after SARS-2-OS protein in vitro stimulation. (N) The correlation between the frequency of CD25⁺ NK cells and the frequency of Ki67⁺ NK cells at 4 weeks after the third dose after SARS-2-OS protein in vitro stimulation. The black hollow circle indicates that PBMCs was only cultured in the medium, while the blue solid circle indicates that PBMCs received SARS-2-OS protein stimulation for 18 h. ^*P <0.05, ^†P <0.01, ^‡P <0.001. IFN: Interferon; MFI: Mean fluorescence intensity; NK: Natural killer; PBMCs: Peripheral blood mononuclear cells; SARS-2-OS: SARS-CoV-2 Omicron Spike.

Phenotype and function of NK cells were altered in PLWH

Next, we compared the differences in NK cell phenotype and function between PLWH and HCs at 0–12 weeks after the third dose of vaccine. Given that SARS-2-OS protein stimulation did not significantly affect the frequency of NK cells and the expression of NKG2A and NKG2C in NK cells, we only analyzed the expression of the aforementioned markers before stimulation. The frequency of CD3^-CD56⁺ NK cells in PLWH was statistically slightly higher than that in HCs at 0–12 weeks after the third dose of vaccine; however, the difference was not statistically significant [Figure 6A]. Similarly, there was no significant difference in the frequency of the CD56^bright NK cells between PLWH and HCs [Figure 6B]. The percentage of CD56^dimCD57^– subset in PLWH was lower than that in HCs, but there were statistically significant differences only at 2 weeks and 12 weeks after the third dose [Figure 6C], the proportion of CD56^dimCD57⁺ subset was higher than that in HCs, and likewise, the difference was statistically significant only at 12 weeks after the third dose of vaccine [Figure 6D]. In addition, the frequencies of NK cells expressing both the inhibitory receptor NKG2A and the activating receptor NKG2C in PLWH were statistically higher than that of HCs, but only the percentage of NKG2C⁺ NK cells showed statistically significant differences at 0–12 weeks after the third dose of vaccine [Figure 6E,F].

Figure 6.

The phenotype and function of NK cells in HIV-infected individuals and healthy subjects at 0–12 weeks after the third dose of vaccine in response to SARS-2-OS protein stimulation in vitro. (A) The frequency of CD3^–CD56⁺ NK cells, (B) the frequency of CD56^bright subset, (C) the frequency of CD56^dimCD57^– subset, (D) the frequency of CD56^dimCD57⁺ subset, (E) the frequency of NKG2A⁺ subset, and (F) the frequency of NKG2C⁺ subset in PLWH and HCs at 0 week, 2 weeks, 4 weeks, and 12 weeks after the third dose with PBMCs were cultured in medium for 18 h, respectively. (G) The frequency of CD16⁺ NK cells, (H) the frequency of CD25⁺ NK cells, (I) the frequency of CD69⁺ NK cells, (J) the frequency of Ki67⁺ NK cells, (K) the frequency of IFN-γ⁺ NK cells, and (L) the frequency of CD107a⁺ NK cells in PLWH and HCs at 0 week, 2 weeks, 4 weeks, and 12 weeks after the third dose before and after PBMCs were stimulated with SARS-2-OS protein for 18 h, respectively. The black hollow circle indicates that the PBMCs of PLWH was only cultured in the medium, while the blue solid circle indicates that the PBMCs of PLWH received SARS-2-OS protein stimulation for 18 h. The black hollow triangle indicates that the PBMCs of HCs was only cultured in the medium, while the blue solid triangle indicates that the PBMCs of HCs received SARS-2-OS protein stimulation for 18 h. ^*P <0.05, ^†P <0.01, ^‡P <0.001. HCs: Healthy controls; NK: Natural killer; PBMCs: Peripheral blood mononuclear cells; PLWH: People live with human immunodeficiency virus; SARS-2-OS: SARS-CoV-2 Omicron Spike.

The proportion of CD16⁺ NK cells in PLWH was significantly higher than that in HCs at 2, 4, and 12 weeks after the third dose with SARS-2-OS protein stimulation in vitro [Figure 6G]. In contrast, the frequency of CD25⁺ NK cells in PLWH was significantly lower than that in HCs at 0, 4, and 12 weeks after the third dose, regardless of whether or not stimulated with SARS-2-OS protein [Figure 6H]. Similarly, the percentage of CD69⁺ NK cells in PLWH was significantly lower than that in HCs at 0 week and 12 weeks after the third dose only being cultured in medium, which was remarkably lower than that in HCs at 0–12 weeks after the third dose of vaccine with SARS-2-OS protein stimulation [Figure 6I]. Furthermore, the percentage of IFN-γ⁺ NK cells in PLWH was significantly lower than that in HCs at 0 week after stimulated with SARS-2-OS protein, and it was lower than that in HCs at 12 weeks after the third dose in the absence of stimulation [Figure 6K]. There was no significant difference in the expression of Ki67 [Figure 6J] and CD107a [Figure 6L] in NK cells between PLWH and HCs at 0–12 weeks after the third dose, whether or not being stimulated with SARS-2-OS protein.

Altogether, these results indicate changes in the composition and receptor profile of NK cell subsets in PLWH after SARS-CoV-2 vaccination, with a significant upregulation of NK cell activation markers and IFN-γ secretion, and a remarkable downregulation of the frequency of CD16⁺ NK cells. However, regardless of whether stimulated by SARS-2-OS protein or not, the expression of NK cell proliferation and degranulation markers in PLWH are comparable to those in HCs after SARS-CoV-2 vaccination.

Discussion

In late 2019, the outbreak of SARS-CoV-2 has caused nearly 768 million infections and 7 million deaths.^[2] NK cells are the first line of defense against viral infections and contribute to early control of viral infections, including herpes, influenza, and SARS-CoV-2. Viral infection or vaccination induces NK cells activation and acquisition of memory-like features, the antiviral response of NK cells is remarkably enhanced upon restimulation with pathogen-associated antigens, thereby contributing to prevention or control infectious disease. Due to immune dysfunction, PLWH have a reduced humoral and cellular immune responses to various vaccines, and an increased hospitalization, severity, and mortality after infection of SARS-CoV-2. A comprehensive understanding of the NK cells response to SARS-CoV-2 vaccine and SARS-2-OS protein stimulation in PLWH and HCs has certain practical implications for the future use of NK cells as an immunotherapy regimen for SARS-CoV-2-related diseases.

In this study, we analyzed the dynamic changes in NK cell phenotype and function, as well as the effect of soluble SARS-CoV-2 Omicron spike glycoprotein on NK cell memory-like responses in PLWH and HCs after the third dose of SARS-CoV-2 inactivated vaccines. The frequency of the CD56^bright subset was comparable between PLWH and HCs. The percentage of the CD56^dimCD57^– subset in PLWH was lower than that of HCs, but the difference was statistically significant only at 2 weeks and 12 weeks after the third dose of vaccine. The proportion of CD56^dimCD57⁺ subset was higher than that of HCs, and the difference was statistically significant only at 12 weeks after the third dose. Tarazona et al^[8] and Frias et al^[9] showed that the percentage of CD56^bright subset in PLWH was comparable with that of HCs, suggesting that HIV infection had no effect on the proportion of CD56^bright subset. However, Hong et al^[10] found that the frequency of CD56^dimCD57^– subsets was significantly reduced in HIV-infected individuals. The percentage of NK cells expressing inhibitory receptor NKG2A and activating receptor NKG2C in PLWH at 0–12 weeks after the third dose was higher than that of HCs; however, only the difference in the frequency of NKG2C⁺ NK cells was statistically significant. Previous studies have revealed that the increased proportion of NKG2C⁺ NK cells in PLWH is mainly caused by cytomegalovirus (CMV) infection.^[11,12] Given that CMV infection is common in both PLWH and HCs, whether the increased proportion of NKG2C⁺ NK in PLWH is associated with CMV infection in this study that needs further investigation.

A growing number of evidence suggests that both inactivated and attenuated live vaccines can induce NK cell activation and proliferation. Jost et al^[13] found that the expression of CD69 and CD25 in CD56^bright NK cells was upregulated at 4 days after receiving the influenza vaccine. Marquardt et al^[14] observed an increase in the frequency of NK cells expressing Ki67, and which peaked at day 10 after the yellow fever vaccination. In addition, Wagstaffe et al^[15] demonstrated that the percentage and proliferation of CD56^bright NK cells increase from day 3 to week 4 after receiving the influenza vaccine. In this study, we found no significant effect of SARS-CoV-2 vaccination on the frequency of NK cells expressing Ki67 in both PLWH and HCs. Nevertheless, stimulation with SARS-2-OS protein significantly increased the percentage of Ki67⁺ NK cells in PLWH at 4 weeks after the third dose, while in HCs, the frequencies of Ki67⁺ NK cells were significantly up-regulated at 4 week and 12 weeks after the third dose. In addition, regardless of whether or not receiving SARS-2-OS protein stimulation, there was no significant difference in the expression of Ki67 on NK cells between PLWH and HCs. These results showed that SARS-CoV-2 vaccination had no effect on the expression of proliferation markers on NK cells both in PLWH and HCs; however, SARS-2-OS protein stimulation could induce a robust NK cell proliferation response. In addition, the frequency of CD25⁺ NK cells in PLWH was significantly up-regulated at 2 weeks after the third dose; however, stimulation with the SARS-2-OS protein did not increase CD25 expression in NK cells of PLWH. On the contrary, SARS-CoV-2 vaccine has no significant effect on the frequency of CD25⁺ NK cells in HCs, but SARS-2-OS protein stimulation can significantly increase the percentage of CD25⁺ NK cells at 4 weeks after the third dose of vaccine. Furthermore, we found that vaccination with SARS-CoV-2 vaccine and SARS-2-OS protein stimulation had no significant effect on the expression of CD69, an early activation marker of NK cells in both PLWH and HCs. Additionally, the frequency of NK cells expressing CD25 and CD69 in HIV-infected individuals was significantly lower than that in healthy subjects. The study by Albarran et al^[16] showed that stimulation of PBMCs with HBsAg for 6 h resulted in significant upregulation of CD25 and CD69 expressions on NK cells in healthy subjects after receiving the HBV vaccine. Horowitz et al^[17] found that after receiving the rabies vaccine, healthy individuals significantly upregulated the expression level of CD69 on NK cells after heat inactivated rabies virus stimulation of PBMCs for 21 h in vitro. However, in this study, we found that SARS-CoV-2 vaccination or SARS-2-OS protein stimulation had no effect on the expression of CD69 on NK cells. The reason for this difference is unclear, but it may reflect the reduced responsiveness of NK cells in HIV-infected individuals to activating stimuli, and further investigation is clearly warranted.

CD16, a low-affinity IgG Fc region receptor III (FcγRIII), is the most effective activating receptor expressed by NK cells. When the Fc region of the IgG antibody on the opsonized cells crosslinked with the CD16 molecule, NK cells were activated by a process termed ADCC, resulting in degranulation and release of perforin and granzyme. It is worth noting that CD16 is the only receptor that can activate NK cells on its own, without any additional activation signal through other receptors.^[18] In this study, we found that the frequency of CD16⁺ NK cells in PLWH gradually decreased after vaccination, reaching its lowest point 2 weeks after the third dose and then gradually increasing. The proportion of CD16⁺ NK cells in PLWH was significantly decreased at 12 weeks after the third dose vaccine as PBMCs were stimulated with SARS-2-OS protein. The frequency of CD16⁺ NK cells was significantly reduced in HCs at 2 weeks and 4 weeks after the third dose with SARS-2-OS protein stimulation in vitro. In addition, the percentage of CD16⁺ NK cell in PLWH was significantly higher than that in HCs at 2 weeks, 4 weeks, and 12 weeks after the third dose. Goodier et al^[19] revealed sustained downregulation of CD16 expression on NK cells in vivo following intramuscular influenza vaccination, and which was associated with influenza-specific plasma antibodies. In addition, in vitro stimulation of NK cells with cytokines such as IL-2, IL-12, and IL-18 can induce NK cell activation while also leading to CD16 downregulation.^[20,21,22] In this study, we only cultured PBMCs in RPMI 1640 medium supplemented as above; therefore, the downregulation of CD16 in NK cells may be related to NK cell activation induced by cytokines secreted by accessory cells. Finally, we found that the frequency of NK cells expressing CD16 was significantly negatively correlated with the percentage of NK cell expressing CD107a and CD25 in PLWH and HCs at different times after the third dose of SARS-CoV-2 vaccine. This is also consistent with the upregulation of activation and cytotoxic markers after NK cell activation, and CD16 may serve as an indirect activation marker for NK cells.

NK cells can directly kill infected target cells through cytolysis, which depends on the release of particles containing perforin and granzyme. Lysosome-associated membrane protein 1 (LAMP1; also known as CD107a), transported to the surface of NK cells after degranulation, can serve as a cytotoxic marker for NK cells.^[23,24] We found that the frequency of NK cells expressing CD107a gradually increased in PLWH after vaccination, and reached a peak level at 2 weeks after the third dose of vaccine, but stimulation with SARS-2-OS protein did not increase the expression of CD107a on NK cells. Similarly, both the frequency of CD107a⁺ of NK cells and CD107a MFI of NK cells peaked at 2 weeks after the third dose, and SARS-2-OS protein stimulation further increased the expression of CD107a on NK cells in HCs. In addition, the frequency of CD107a⁺ of NK cells and CD107a MFI of NK cells in PLWH and HCs were comparable. These studies indicated that SARS-CoV-2 vaccination remarkably enhanced the cytotoxicity of NK cells in both PLWH and HCs, and SARS-2-OS protein stimulation in vitro further enhanced the degranulation of NK cells in HCs but not in PLWH. In contrast, a recent study by Cuapio et al^[25] showed that the total number, frequency, phenotype, and function of NK cells in immune-deficient people (including PLWH and people who received organ transplantation) were comparable with those in HCs after BNT162b2 mRNA SRAS-CoV-2 vaccine inoculation, indicating that mRNA SARS-CoV-2 vaccine has no effect on NK cell response. However, the reasons for this discrepancy are unclear and require further study.

There may be some possible limitations in this study. Firstly, since most healthy individuals have already received their first dose of SARS-CoV-2 vaccine at the time of enrollment, we are unable to collect blood samples from them before the vaccination. Secondly, due to the fact that all HIV-infected individuals have received continuous ART and achieved viral suppression, and the vast majority of participants have CD4 counts greater than 500 cells/μL, we cannot analyze the impact of severe immunodeficiency and HIV virus replication on NK cell response. Finally, due to the limited total number of blood samples, we are unable to perform NK cell sorting and blocking experiments to determine the exact activation mechanism of NK cells.

In conclusion, the phenotype, receptor profile and function of NK cell subsets in HIV-infected individuals were altered after three dose of vaccine. The SARS-CoV-2 vaccine elicited activation, proliferation, and degranulation of NK cells in both PLWH and HCs. We demonstrated that three doses of SARS-CoV-2 inactivated vaccine elicited a durable NK cell response, which has certain implications for the future use of NK cells for immunotherapy of COVID-19-related diseases.

Funding

This project was supported by grants from the National Natural Science Foundation of China (Nos. 82272319 and 82072271), Beijing Natural Science Foundation (No. L222068), the High-Level Public Health Specialized Talents Project of Beijing Municipal Health Commission (Nos. 2022-2-018 and 2022-1-007), the Climbing the peak (Dengfeng) Talent Training Program of Beijing Hospitals Authority (No. DFL20191701), the Beijing Health Technologies Promotion Program (No. BHTPP202002), Scientific Research Project of Beijing Youan Hospital-CCMU 2022 (No. BJYAYY-YN-2022-18), and Beijing Key Laboratory for HIV/AIDS Research (No. BZ0089).

Conflicts of interest

None.