HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

doi:10.1038/s41467-023-36109-8

. 2023 Feb 2;14(1):575.

doi: 10.1038/s41467-023-36109-8.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

Massimiliano Bissa¹, Sohyoung Kim², Veronica Galli³, Slim Fourati^{4

5}, Sarkis Sarkis³, Anush Arakelyan⁶, Isabela Silva de Castro³, Mohammad Arif Rahman³, Saori Fujiwara², Monica Vaccari^{3

7}, Jeffrey A Tomalka^{4

5}, James D Stamos³, Luca Schifanella³, Giacomo Gorini³, Ramona Moles³, Anna Gutowska³, Guido Ferrari⁸, Alexei Lobanov⁹, David C Montefiori⁸, George W Nelson⁹, Margaret C Cam⁹, Marita Chakhtoura¹⁰, Elias K Haddad¹⁰, Melvin N Doster³, Katherine McKinnon¹¹, Sophia Brown^{3

11}, David J Venzon¹², Hyoyoung Choo-Wosoba¹², Matthew W Breed¹³, Kristin E Killoran¹³, Joshua Kramer¹³, Leonid Margolis⁶, Rafick P Sekaly^{4

5}, Gordon L Hager², Genoveffa Franchini¹⁴

Affiliations

¹ Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, MD, USA. massimiliano.bissa@nih.gov.
² Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
³ Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, MD, USA.
⁴ Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.
⁵ Department of Pathology, Emory University, Atlanta, GA, USA.
⁶ Section on Intercellular Interactions, Eunice Kennedy-Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
⁷ Tulane National Primate Research Center, Tulane University, Covington, LA, USA.
⁸ Division of Surgical Sciences, Duke University School of Medicine, Durham, NC, USA.
⁹ Collaborative Bioinformatics Resource, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
¹⁰ Department of Medicine, Drexel University College of Medicine, Philadelphia, PA, USA.
¹¹ Vaccine Branch Flow Cytometry Core, National Cancer Institute, Bethesda, MD, USA.
¹² Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
¹³ Laboratory Animal Sciences Program, Leidos Biomedical Research Inc., Frederick National Laboratory, Frederick, MD, USA.
¹⁴ Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, MD, USA. franchig@mail.nih.gov.

PMID: 36732510
PMCID: PMC9894672
DOI: 10.1038/s41467-023-36109-8

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

Massimiliano Bissa et al. Nat Commun. 2023.

. 2023 Feb 2;14(1):575.

doi: 10.1038/s41467-023-36109-8.

Authors

Affiliations

¹ Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, MD, USA. massimiliano.bissa@nih.gov.
² Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
³ Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, MD, USA.
⁴ Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.
⁵ Department of Pathology, Emory University, Atlanta, GA, USA.
⁶ Section on Intercellular Interactions, Eunice Kennedy-Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
⁷ Tulane National Primate Research Center, Tulane University, Covington, LA, USA.
⁸ Division of Surgical Sciences, Duke University School of Medicine, Durham, NC, USA.
⁹ Collaborative Bioinformatics Resource, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
¹⁰ Department of Medicine, Drexel University College of Medicine, Philadelphia, PA, USA.
¹¹ Vaccine Branch Flow Cytometry Core, National Cancer Institute, Bethesda, MD, USA.
¹² Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
¹³ Laboratory Animal Sciences Program, Leidos Biomedical Research Inc., Frederick National Laboratory, Frederick, MD, USA.
¹⁴ Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, MD, USA. franchig@mail.nih.gov.

PMID: 36732510
PMCID: PMC9894672
DOI: 10.1038/s41467-023-36109-8

Abstract

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14⁺ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14⁺ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

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Conflict of interest statement

The authors declare no financial and non-financial competing interests. The US government has filed the patent, NIH (DHHS) Ref. No. E-160-2018-0-EP-05 on the HIV ΔV1-envelope immunogens.

Figures

**Fig. 1. Study design, vaccine efficacy, and monocytes.**
a, b Age of vaccinated (a; p = 0.00000002) and control (b; p = 0.0000000017) animals. c Schematic study design of Study 1 with immunization schedule (weeks 0–12) and SIV_mac251 challenges (weeks 17–27). d, e SIV_mac251 acquisition. The number of intravaginal exposures before infection was assessed in (d) n = 13 young and (e) n = 17 old animals relative to their n = 27 young and n = 11 old control animals (Log-rank Mantel-Cox test). f, g Log₁₀ of SIV-DNA copies in vaginal mucosa at 2–3 weeks after infection in (f) n = 7 vaccinated and n = 15 control young animals; and (g) n = 11 vaccinated and n = 9 control old animals. h Correlation between the frequency of classical monocytes (CD14⁺CD16⁻HLA-DR⁺ in live cells) at week 13 and the time of acquisition (TOA) in n = 13 young animals. i Frequency of classical monocytes in n = 13 young and n = 11 old vaccinated animals (week 13). j Classical monocytes expressing CCR2 (CCR2⁺ in CD14⁺CD16⁻ cells) in n = 13 young and n = 13 old vaccinated animals (week 13). k Serum CCL2 levels (pg/ml) in n = 13 young and n = 17 old vaccinated animals (average weeks 10 and 14; p = 0.0000001). l Correlation between the arginase activity measured in plasma at week 13 and the average of the serum CCL2 levels (pg/ml) at weeks 10 and 14 in n = 13 young vaccinated animals. m, n Correlations between (m) the average of adjusted specific ADCC killing of SIV_mac251-infected cells at week 14 assessed at different plasma dilutions and the TOA, or (n) the average of the serum CCL2 levels (pg/ml) at weeks 10 and 14 in n = 13 young vaccinated animals. o Correlation between the frequency of intermediate monocytes (CD14⁺CD16⁺HLA-DR⁺ in live cells) at week 13 and the TOA in n = 13 young animals. Comparisons: a, b, f, g, i–k Two-tailed Mann–Whitney t-test with median. Correlation analyses: h, l–o two-tailed Spearman correlation test and simple linear regression. Displayed p values are unadjusted. Source data are provided in the Source Data file.

**Fig. 2. Transcriptome and extracellular vesicular microRNAs.**
a Multidimensional scaling plot summarizing gene expression. Different responses to vaccination identified in n = 13 young and n = 17 old animals. Differences were present between baseline and 24 h following vaccination (first dimension, Wilcoxon test, p = 3.18 × 10⁻¹¹) and between young and old groups (second dimension, Mann–Whitney, p = 8.77 × 10⁻⁶). b, c Expression levels of genes in BioAge modules M9 and M16 associated with age and risk of SIV_mac251 acquisition in n = 13 young and n = 17 old animals (week 12 + 24 h). The median, 95% CI, and first and third quartiles are displayed. d, e Expression of *ZC3H7A* gene at baseline and 24 h following vaccination in (d) n = 13 young and (e; p = 0.00003) n = 17 old animals. f, g Correlations between the expression of *ZC3H7A* at 24 h following vaccination and the Time of Acquisition (TOA) in (f) n = 13 young and (g) n = 17 old vaccinated animals. h, i, l Correlations between the expression of h miR-139-5p, i miR-29b-1-5p, or l miR-98 in EVs (week 13) and TOA in n = 12 young animals. j, k, m Normalized expression of j miR-139-5p, k miR-29b-1-5p, and m miR-98 in n = 7 young animals at baseline and n = 12 young vaccinated animals at week 13. n, o Expression of Phosphodiesterase-4D (*PDE4D*) at baseline and 24 h following the last boost in the (n) n = 13 young and (o; p = 0.00066) n = 17 old animals. Comparisons: d, e, n, o two-tailed Wilcoxon signed rank or j, k, m two-tailed Mann–Whitney tests with median. Correlation analyses: f–i, l two-tailed Spearman correlation tests and simple linear regression. Displayed p values are unadjusted. Source data are provided in the Source Data file and at GEO Series accession numbers GSE188901 and GSE188575.

**Fig. 3. Chromatin accessibility to *CREB1*. V2 specific ADCC and risk of SIV acquisition.**
a Schematic study design with time of immunization (weeks 0–12) or SIV_mac251 challenges (weeks 17–27) and time of collection of ATAC- and RNA-seq samples (black triangle). b SIV_mac251 acquisition. The number of intravaginal exposures before viral acquisition was assessed in n = 12 young animals relative to n = 37 historic controls (Log-rank Mantel-Cox test). c Two-tailed Spearman correlation and simple linear regression between V2-specific ADCC at week 14 and time of acquisition (TOA) in n = 12 animals. d Volcano plot showing in red ATAC-sites with significantly (FDR < 0.05) different accessibility at baseline and week 13 and with absolute fold-change higher than 1.5 (Log₂ = 0.585). Reduced accessibility: 641 sites, Increased: 4026. Blue dots: sites with significantly different accessibility, but lower absolute fold-change. Green dots: sites with not significantly different accessibility, but absolute fold-change higher than 1.5. Gray dots: sites with not significantly different accessibility, but low absolute fold-change. Comparisons performed by two-tailed paired analyses. e Two-tailed Spearman correlation and simple linear regression of variation between baseline and week 13 of the intensity of ATAC site downstream *CREB1* TSS accessibility (chr12:95218442-95218735) identified in Supplementary Fig. S6b with TOA in n = 11 vaccinated animals (week 13). f Visualization of the ATAC site downstream *CREB1* TSS (chr12:95218442-95218735) whose variation between baseline and week 13 correlated with TOA. The ATAC site of each animal is shown at baseline and week 13 for better comparison (n = 11 animals). Animals that showed an increase in accessibility following vaccination are indicated by the red box. Animals are colored based on TOA, and those with TOA > 8 are framed. Displayed p values are unadjusted. Source data are provided in the Source Data file and at GEO Series accession numbers GSE188879 and GSE189032.

**Fig. 4. *CREB1* pathway activation and ADCC.**
a Heatmap representations of the log₂ fold-change (logFC) expression (week 13/baseline) in CD14⁺ cells (n = 12 animals) of 20 genes involved in the cellular response to cAMP identified by Tomalka et al. and their correlation with V2-specific ADCC. b Heatmap representations of the log₂ fold-change expression (week 13/baseline) in CD14⁺ cells (n = 12 animals) of 16 genes involved in Gene Ontology Biological Process (GOBP) related to the cAMP-mediated signaling and their correlation with V2-specific ADCC. In a, b, the V2-specific ADCC response is reported on the top row. c Relative expression of miR-139-5p in plasma collected at baseline and week 13 in n = 12 animal (two-tailed Wilcoxon signed rank). Displayed p values are unadjusted. Source data are provided in the Source Data file and at GEO Series accession number GSE189032.

**Fig. 5. Efferocytosis and DC-10 in Study 2.**
a Correlation between the frequency of CD14⁺ efferocytes cocultured for 24 h and Time of Acquisition (TOA) in n = 9 vaccinated animals (week 13). b, c Correlation between the change from baseline to week 14 in the frequency of DC-10 cells and the frequency of CD14⁺ efferocytes cocultured for (b) 24 h in n = 9 vaccinated animals (week 13) or (c) the TOA in n = 11 vaccinated animals. Gating strategy: high SSC/single cells/Live/CD45⁺/Lin⁻(CD3⁻CD20⁻)/HLA-DR⁺/ CD1c⁻/CD11b⁺/CD11c⁺/CD14⁺CD16⁺/CD163⁺/CD141⁺/CD1a⁻. The frequency of DC-10 cells was identified as the frequency of CD1a⁻ cells in CD11b⁺ cells. Correlation analyses: a–c Two-tailed Spearman correlation test and simple linear regression. Displayed p values are unadjusted. Source data are provided in the Source Data file.

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References

1. Rerks-Ngarm S, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 2009;361:2209–2220. - PubMed
1. Vaccari M, et al. Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition. Nat. Med. 2016;22:762–770. - PMC - PubMed
1. Vaccari M, et al. HIV vaccine candidate activation of hypoxia and the inflammasome in CD14(+) monocytes is associated with a decreased risk of SIVmac251 acquisition. Nat. Med. 2018;24:847–856. - PMC - PubMed
1. Gorini G, et al. Engagement of monocytes, NK cells, and CD4+ Th1 cells by ALVAC-SIV vaccination results in a decreased risk of SIVmac251 vaginal acquisition. PLoS Pathog. 2020;16:e1008377. - PMC - PubMed
1. Silva de Castro I, et al. Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates. iScience. 2021;24:102047. - PMC - PubMed

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[1] Rerks-Ngarm S, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 2009;361:2209–2220. - PubMed

[2] Rerks-Ngarm S, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 2009;361:2209–2220. - PubMed

[3] Vaccari M, et al. Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition. Nat. Med. 2016;22:762–770. - PMC - PubMed

[4] Vaccari M, et al. Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition. Nat. Med. 2016;22:762–770. - PMC - PubMed

[5] Vaccari M, et al. HIV vaccine candidate activation of hypoxia and the inflammasome in CD14(+) monocytes is associated with a decreased risk of SIVmac251 acquisition. Nat. Med. 2018;24:847–856. - PMC - PubMed

[6] Vaccari M, et al. HIV vaccine candidate activation of hypoxia and the inflammasome in CD14(+) monocytes is associated with a decreased risk of SIVmac251 acquisition. Nat. Med. 2018;24:847–856. - PMC - PubMed

[7] Gorini G, et al. Engagement of monocytes, NK cells, and CD4+ Th1 cells by ALVAC-SIV vaccination results in a decreased risk of SIVmac251 vaginal acquisition. PLoS Pathog. 2020;16:e1008377. - PMC - PubMed

[8] Gorini G, et al. Engagement of monocytes, NK cells, and CD4+ Th1 cells by ALVAC-SIV vaccination results in a decreased risk of SIVmac251 vaginal acquisition. PLoS Pathog. 2020;16:e1008377. - PMC - PubMed

[9] Silva de Castro I, et al. Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates. iScience. 2021;24:102047. - PMC - PubMed

[10] Silva de Castro I, et al. Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates. iScience. 2021;24:102047. - PMC - PubMed

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HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

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HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

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