Vaccine induced mucosal and systemic memory NK/ILCs elicit decreased risk of SIV/SHIV acquisition

Abstract

SIV and HIV-based envelope V1-deleted (ΔV1) vaccines, delivered systemically by the DNA/ALVAC/gp120 platform, decrease the risk of mucosal SIV or SHIV acquisition more effectively than V1-replete vaccines. Here we investigated the induction of mucosal and systemic memory-like NK cells as well as antigen-reactive ILC response by DNA/ALVAC/gp120-based vaccination and their role against SIV/SHIV infection. ΔV1 HIV vaccination elicited a higher level of mucosal TNF-α⁺ and CD107⁺ memory-like NK cells than V1-replete vaccination, suggesting immunogen dependence. Mucosal memory-like NK cells, systemic granzyme B⁺ memory NK cells, and vaccine-induced mucosal envelope antigen-reactive IL-17⁺ NKp44⁺ ILCs, IL-17⁺ ILC3s, and IL-13⁺ ILC2 subsets were linked to a lower risk of virus acquisition. Additionally, mucosal memory-like NK cells and mucosal env-reactive IFN-γ⁺ ILC1s and env- reactive IL-13⁺ ILC2 subsets correlated with viral load control. We further observed a positive correlation between post-vaccination systemic and mucosal memory-like NK cells, suggesting vaccination enhances the presence of these cells in both compartments. Mucosal and systemic memory-like NK cells positively correlated with V2-specific ADCC responses, a reproducible correlate of reduced risk of SIV/HIV infection. In contrast, an increased risk was associated with the level of mucosal PMA/Ionomycin-induced IFN-γ⁺ and CD107⁺ NKG2A^-NKp44^- ILCs. Plasma proteomic analyses demonstrated that suppression of mucosal memory-like NK cells was linked to the level of CCL-19, LT-α, TNFSF-12, and IL-15, suppression of systemic env-reactive granzyme B⁺ memory-like NK cells was associated with the level of OLR1, CCL-3, and OSM, and suppression of IL-17⁺ ILCs immunity was correlated with the level of IL-6 and CXCL-9. In contrast, FLT3 ligand was associated with promotion of protective mucosal env-reactive IL-17⁺ responses. These findings emphasize the importance of mucosal memory-like NK cell and envelope- reactive ILC responses for protection against mucosal SIV/SHIV acquisition.

Keywords: SIV/SHIV; V2-specific ADCC; antigen-reactive ILCs; cytokines; innate memory; memory-like NK cells; trained immunity; vaccine (DNA/ALVAC/gp120).

Figure 1

Immunization regimen and identification of memory-like NK cell responses in mucosa. (A) A group of 18 female, rhesus macaques (ΔV1 SIV vaccine) was primed with DNA-SIVgp160 ΔV1+SIV_mac239 gag and boosted with ALVAC-SIV encoding env, gag, and pol and ALVAC-SIV+ΔV1 gp120 protein in alum hydroxide at the indicated timepoints. Beginning at week 17, protective efficacy against SIV_mac251 was assessed by subjecting all animals to up to 14 weekly intravaginal viral exposures (arrows) until infection was confirmed. The animals were challenged intravaginally weekly with 1 ml of a SIV_mac251 stock (23, 24, 69, 70) containing 800 median tissue culture infectious doses (TCID50). (B) A subset of 12 male rhesus macaques (WT HIV vaccine) was primed with DNA-HIVgp160+HIV gag and boosted with ALVAC-HIV encoding env, gag, and pol and ALVAC-HIV+gp120 protein in alum hydroxide at the indicated timepoints. Another subset of 12 male rhesus macaques (ΔV1 HIV vaccine) was primed with DNA-HIVgp160 ΔV1+HIV gag and boosted with ALVAC-HIV encoding env, gag, and pol and ALVAC-HIV+ΔV1 gp120 protein in alum hydroxide at the indicated timepoints. Beginning at week 21, protective efficacy against SHIV was assessed by subjecting all animals to up to 11 weekly intrarectal viral exposures (arrows) until infection was confirmed. The animals were challenged intrarectally weekly with 1 ml of a SHIV_{157(QNE)Y173H} (26, 64) diluted 1:10,000 from stock. (C) Gating strategy of memory-like NK cells. (D, E) Evaluation of (D) memory-like NK cells and (E) env-reactive IFN-γ⁺ memory-like NK cells over the course of vaccination in the female macaques. (F) Correlation of memory-like NK cells at week 13 in the female macaques with number of challenges. (G) Evaluation of memory-like NK cells over the course of vaccination in the subsets of male macaques. (H) Correlation of memory-like NK cells at week 13 in all 24 male macaques with number of SHIV challenges. (I, J) Evaluation of (I) PMA/Ionomycin -induced CD107⁺ memory-like NK cells and (J) PMA/Ionomycin -induced TNF-α⁺ memory-like NK cells over the course of vaccination in the subsets of male macaques. (K) Correlation of memory-like NK cells with V2-specific ADCC. (L, M) Correlation of env-reactive IFN-γ⁺ memory-like NK cells with (L) CCR5^-α4β7⁺Th17 cells and (M) CCR5^-α4β7^-Th17 cells in blood. (N) Correlation of memory-like NK cells with VL at 1 wpi. (O) Correlation of env-reactive IFN-γ⁺ memory-like NK cells with VL at 8 wpi. Data shown in (C, D, G, I, J) were analyzed with Wilcoxon signed-rank test or Mann-Whitney test. Data shown in (E, H, K-O) were analyzed by the Spearman correlation test. Horizontal and vertical bars denote mean and SD, respectively. Violin plot vertical bars denote median and quartiles. Here, black, red and blue symbols represent ΔV1 SIV vaccinated female macaques, WT HIV vaccinated male macaques and ΔV1 HIV vaccinated male macaques, respectively.

Figure 2

Evaluation of antigen-reactive ILC responses in mucosa. (A) Gating strategy of ILCs. (B-H) Evaluation of (B) ILC1, (C) ILC2, (D) ILC3, (E) env-reactive IFN-γ⁺ ILC1, (F) env-reactive IL-13⁺ ILC2, (G) env-reactive IL-17⁺ ILC3, over the course of vaccination of female macaques and (H) PMA/Ionomycin -induced IL-17⁺ ILC3 over the course of vaccination of male macaques. (I-K) Correlation of (I) env-reactive IL-13⁺ ILC2, (J) env-reactive IL-17⁺ ILC3, and (K) PMA/Ionomycin -induced IL-17⁺ ILC3 with number of challenges. (L-O) Correlation of (L) ILC1, (M) env-reactive IFN-g⁺ ILC1, (N) env-reactive IL-13⁺ ILC2, and (O) PMA/Ionomycin -induced IL-13⁺ ILC2 with VL. Data shown in (B-H) were analyzed with Wilcoxon signed-rank test or Mann-Whitney test. Data shown in (I-O) were analyzed by the Spearman correlation test. Horizontal and vertical bars denote mean and SD. Violin plot vertical bars denote median and quartiles. Here, black, red and blue symbols represent ΔV1 SIV vaccinated female macaques, WT HIV vaccinated male macaques and ΔV1 HIV vaccinated male macaques, respectively.

Figure 3

Evaluation of NK/ILC responses in mucosa. (A) Gating strategy of NK/ILCs. (B, C) Evaluation of (B) NKG2A⁺ NK cells and (C) NKp44⁺ ILCs over the course of vaccination of female macaques. (D, E) Correlation of NKp44⁺ ILCs with number of challenges in the (D) female and (E) male macaques. (F, G) Correlation of NKp44⁺ ILCs with (F) V2-specific ADCC and (G) vaccine induced Th2 cells. (H) Evaluation of env-reactive IL-17⁺ NKp44⁺ ILCs over the course of vaccination of female macaques. (I-K) Correlation of env-reactive IL-17⁺ NKp44⁺ ILCs with (H) number of challenges, (I) V2-specific ADCC and (K) vaccine induced Th17 cells in the female macaques. (L) Evaluation of NKG2A^- NKp44^- ILCs over the course of vaccination of female macaques. (M, N) Correlation of env-reactive IFN-γ⁺ NKG2A^- NKp44^- ILCs with (M) number of challenges and (N) VL in female macaques. (O) Correlation of PMA/Ionomycin -induced IFN-γ⁺ NKG2A^- NKp44^- ILCs with VL in female macaques. Data shown in (B, C, H, L) were analyzed with Wilcoxon signed-rank test. Data shown in (D-G, I-K, M-O) were analyzed by the Spearman correlation test. Horizontal and vertical bars denote mean and SD, respectively. Here, gray, red and blue symbols represent ΔV1 SIV vaccinated female macaques, WT HIV vaccinated male macaques and ΔV1 HIV vaccinated male macaques, respectively.

Figure 4

Evaluation of systemic NK/ILC responses in female macaques. (A, B) Gating strategy of NK/ILCs. (C) Evaluation of memory-like NK cells over the course of vaccination of female macaques. (D) Correlation of systemic memory-like NK cells with number of challenges. (E) Correlation of systemic and mucosal memory-like NK cells. (F) Correlation of systemic memory-like NK cells with V2-specific ADCC. (G, H) Evaluation of (G) env-reactive GranB⁺ memory-like NK cells and (H) env-reactive IFN-γ⁺ memory-like NK cells over the course of vaccination of female macaques. (I) Correlation of systemic env-reactive GranB⁺ memory-like NK cells with number of challenges. (J, K) Correlation of (J) systemic memory-like NK cells and (K) NK cells with VL. (L) Evaluation of ILC1 over the course of vaccination of female macaques. (M, N) Correlation of (K) ILC1 and (L) ILC2 with number of challenges. (O, P) Correlation of (O) env-reactive IFN-γ⁺ ILC1 and (P) env-reactive IL-17⁺ ILC3 with VL. Violin plot data shown in (C, G, H, L) were analyzed with Wilcoxon signed-rank test. Data shown in (D-F, I-K, M-P) were analyzed by the Spearman correlation test. Violin plot vertical bars denote median and quartiles. Here, black symbols represent ΔV1 SIV vaccinated female macaques.

Figure 5

Correlation of plasma cytokines/chemokines with mucosal/systemic immune responses. (A) Radar plots comparing the level of 36 cytokines/chemokines in the plasma of SIV ΔV1 vaccinated animals at 24 hrs after last immunization (Week12 + 24hrs) and of HIV vaccinated animals 1 week after last immunization (Week13). (B-E) Correlation of mucosal memory-like NK cell frequency with (B) CCL-19, (C) LT-α, (D) TNFSF-12, and (E) IL-15. (F-H) Correlation of systemic env-reactive GranB⁺ memory-like NK cell frequency with (F) OLR-1, (G) CCL-3, and (H) OSM. (I, J) Correlation of IL-6 with (I) mucosal env-reactive IL-17⁺ ILC3 and (J) mucosal env-reactive IFN-γ⁺ NKG2A^-NKP44^- ILCs. (K, L) Correlation of FLT3 ligand with (K) mucosal env-reactive IL-17⁺ NKp44⁺ ILCs and (L) mucosal env-reactive IL-17⁺ ILC3. Data for female macaques are shown in panels (B, C, F-L), and data for male macaques shown in panels (D, E). Data shown in (B-L) were analyzed by the Spearman correlation test. The radar plot represents the mean value of cytokine responses. Here, black/gray, red and blue symbols represent ΔV1 SIV vaccinated female macaques, WT HIV vaccinated male macaques and ΔV1 HIV vaccinated male macaques, respectively.

Figure 6

Protective role of vaccine induced mucosal/systemic NK/ILC in SIV/SHIV infection. Administration of vaccine increases different NK/ILC populations in the mucosa as well as in blood. Mucosal env-reactive IL-17⁺ NKp44⁺ ILCs, env-reactive IL-17⁺ ILC3, env-reactive IL-13⁺ ILC2, NKp44⁺ ILCs, and memory-like NK cells, as well as systemic memory-like NK cells, ILC1 and ILC2 showed a protective role against SIV/SHIV infection. IL-15, CCL-19, LT-α, and TNFSF-12 cytokines suppress the mucosal memory-like NK cells and OLR1, CCL3 and OSM suppress systemic GranB⁺ memory-like NK cells. IL-6 suppresses protective mucosal env-reactive IL-17⁺ NKp44⁺ ILCs responses. FLT3 ligand facilitate protective mucosal env-reactive IL-17⁺ NKp44⁺ ILCs and env-reactive IL-17⁺ ILC3 responses. This figure was created with BioRender.com.