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. 2024 Sep 26;27(11):111055.
doi: 10.1016/j.isci.2024.111055. eCollection 2024 Nov 15.

The BNT162b2 mRNA vaccine demonstrates reduced age-associated TH1 support in vitro and in vivo

Byron Brook  1   2 , Abhinav Kumar Checkervarty  1   3   4 , Soumik Barman  1   2 , Cali Sweitzer  1 , Anna-Nicole Bosco  1 , Amy C Sherman  1   5 , Lindsey R Baden  5 , Elena Morrocchi  1   2   6 , Guzman Sanchez-Schmitz  1   2 , Paolo Palma  6   7 , Etsuro Nanishi  1   2 , Timothy R O'Meara  1 , Marisa E McGrath  8 , Matthew B Frieman  8 , Dheeraj Soni  9 , Simon D van Haren  1   2 , Al Ozonoff  1   2   10 , Joann Diray-Arce  1   2 , Hanno Steen  1   11 , David J Dowling  1   2 , Ofer Levy  1   2   10
Affiliations

The BNT162b2 mRNA vaccine demonstrates reduced age-associated TH1 support in vitro and in vivo

Byron Brook et al. iScience. .

Abstract

mRNA vaccines demonstrate impaired immunogenicity and durability in vulnerable older populations. We hypothesized that human in vitro modeling and proteomics could elucidate age-specific mRNA vaccine actions. BNT162b2-stimulation changed the plasma proteome of blood samples from young (18-50Y) and older adult (≥60Y) participants, assessed by mass spectrometry, proximity extension assay, and multiplex. Young adult up-regulation (e.g., PSMC6, CPN1) contrasted reduced induction in older adults (e.g., TPM4, APOF, APOC2, CPN1, PI16). 30-85% lower TH1-polarizing cytokines and chemokines were induced in elderly blood (e.g., IFNγ, CXCL10). Analytes lower in older adult samples included human in vivo mRNA immunogenicity biomarkers (e.g., IFNγ, CXCL10, CCL4, IL-1RA). BNT162b2 also demonstrated reduced CD4+ TH1 responses in aged vs. young adult mice. Our study demonstrates the utility of human in vitro platforms modeling age-specific mRNA vaccine immunogenicity, highlights impaired support of TH1 polarization in older adults, and provides a rationale for precision mRNA vaccine adjuvantation to induce greater immunogenicity.

Keywords: Geriatrics; Health sciences; Immunity; Immunology; Proteomics.

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Conflict of interest statement

O.L. has served as a consultant to GlaxoSmithKline (GSK) and Hillevax. M.B.F. serves on the scientific advisory board of Aikido Pharma and has collaborative research agreements with Novavax, AstraZeneca, Regeneron, and Irazu Bio. B.B., E.N., T.R.O., D.S., S.H., O.L., and D.J.D. are named inventors on vaccine adjuvant patent(s). O.L., G.S.S., and D.J.D. are named inventors on patents related to human in vitro modeling of vaccine responses. O.L. and G.S.S. are recipients of a sponsored research agreement with GSK. D.J.D is on the scientific advisory board of EdJen BioTech and serves as a consultant with Merck Research Laboratories/Merck Sharp & Dohme Corp. (a subsidiary of Merck & Co., Inc.). O.L. and D.J.D. are co-founders of and advisors to Ovax, Inc. ACS and LRB are involved in HIV, COVID, and other vaccine clinical trials conducted in collaboration with the NIH, HIV Vaccine Trials Network (HVTN), COVID Vaccine Prevention Network (CoVPN), International AIDS Vaccine Initiative (IAVI), Crucell/Janssen, Moderna, and Sanofi. These commercial or financial relationships are unrelated to the current study. The participating Precision Vaccines Program (PVP) laboratories were supported in part, by U.S. National Institutes of Health (NIH)/National Institutes of Allergy and Infectious Diseases (NIAID) awards, including Human Immunology Project Consortium award U19 AI118608, Adjuvant Discovery (HHSN272201400052C) and Development (HHSN272201800047C) Program Contracts to O.L.; Adjuvant Discovery Program contract (75N93019C00044) to O.L. and D.J.D as well as NIH grant (1R21AI137932-01A1) to D.J.D. O.L. is also funded by an award from the Coalition for Epidemic Preparedness Innovations (CEPI), via the International Network of Special Immunization Services (INSIS). The PVP is supported, in part, by the BCH Department of Pediatrics and philanthropy via the BCH Trust, including from the Barry Family and the Boston Investment Council. A.K.C. was supported by the Friedman Award for Scholars in Health, the University of British Columbia, and Mitacs Accelerate Canada.

Figures

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Graphical abstract
Figure 1
Figure 1
mRNA vaccine induced greater in vitro inflammatory protein release in adult vs. elder whole blood assay Adult and elder whole blood stimulated with vehicle control (RPMI) or BNT162b2 had supernatants characterized by LC/MS (A-B) and PEA (C-D). GEEGLM evaluation testing concentration-dependent down- (purple) and up-regulated (orange) protein expression is presented against log2FC of BNT162b2-stimulated samples over matching control, from (A) adult and (B) elder participant samples. Log2FC of 2μg/ml BNT162b2’s mRNA over matching vehicle control display PEA-quantified (C) adult and (D) elder responses. Horizontal dotted lines represent -log10(0.05). For (A-B) n = 10 to 14; for (C-D) n = 4 to 5. Statistical significance was determined by (A-B) GEEGLM, and (C-D) paired moderated T-test.
Figure 2
Figure 2
Lower BNT162b2-induced inflammatory response in elder vs. adult whole blood assayed in vitro (A) DEPs from BNT162b2-stimulated samples against paired vehicle controls (RPMI) were predominantly nonoverlapping between age groups. Comparing BNT162b2-stimulated adult and elder samples identified upregulation in adult participants with analyte quantification by (B) LC/MS-proteomics or (C) PEA-proteomics. (D) Advancing age (years, Y) negatively correlated with normalized protein expression (NPX) in BNT162b2 (BNT)-induced CCL4 (Spearman’s correlation analysis p = 0.04), with 95% confidence interval graphed in gray. (B-C) Horizontal dotted lines represent -log10(0.05). For (A-B) n = 10 to 14; for (C-D) n = 4 to 5. Statistical significance was determined by (B) GEEGLM, (C) paired moderated T-test, and (D) Spearman’s correlation.
Figure 3
Figure 3
BNT162b2 induced concentration- and age-dependent cytokine and chemokine production in in vitro human whole blood assay Multiplex quantification of secreted analytes identified BNT162b2-induced responses compared to vehicle control (RPMI). (A) mRNA vaccine concentration-dependent induction of IL-6, CXCL8, TNF, and IFNγ was noted in adults. (B) Fold Change (FC) baseline standardization of stimulated over matching vehicle control demonstrated greater production of certain analytes such as CXCL10, IL-1RA, and IFNγ in adult (solid red line) vs. elder (dashed blue line) blood. (C) Volcano plot of analytes with greater BNT162b2 fold-induced stimulation in adults than in elders, with circles representing 0.2 μg/mL, squares 0.67 μg/mL, and diamonds 2.0 μg/mL of mRNA encapsulated in BNT162b2. Non-filled, crossed points represent markers associated with high vaccine responsiveness. The dotted line represents significance, with points annotated above 1.3 -log10(p-value). For (A-C) n = 12 to 14. Boxplots display the median, interquartile range (IQR), with the identification of the furthest values from the median not exceeding 1.5 × IQR. Statistical significance was determined by Shapiro-Wilk then (A) paired Wilcoxon rank-sum test, (B, C) 1-sided unpaired T tests on log-transformed fold-change, with p-values annotated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 4
Figure 4
Impaired BNT162b2-induced TH1-polarizing cytokine production in human older vs. young adults’ blood Radar plots displaying log10-transformed, FC averages of multiplex-quantified analytes per spoke, separating adult (orange-red) and elder (blue-teal) participants. Stimulation with BNT162b2 (BNT) encapsulated mRNA weights of (A) 0.2 μg/mL, (B) 0.67 μg/mL, and (C) 2 μg/mL had TH1-polarizing analytes (per Table S3) significantly induced (one-sided T-tests hypothesizing induction compared to vehicle control, color-coded orange adult and teal elder asterisks presented above each analyte). GEEGLM analyses evaluating the interaction of age and induction of TH1 polarizing analytes demonstrated 7.2% less TH1-polarizing cytokine production in elder participant samples compared to adult samples (p = 0.027). For (A-C), n = 12 to 14. Significance displays one-sided unpaired T-tests compared to vehicle control, with p-values annotated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 5
Figure 5
Reduced humoral and TH1 cellular immunogenicity of BNT162b2 vaccine in aged mice BNT162b2-immunized adult and aged mice had humoral immunity evaluated on Day 42 post-prime immunization for receptor binding domain (RBD) responsivity. (A) Total anti-RBD IgG was significantly induced, but with significantly lower Ab titers in aged mice. With FC normalization, aged mice additionally had (B) lower CD4+ T cell IFNγ+ and TNF+ (TH1) positivity, and (C) lower CD8+ T cell TNF+ positivity. For (A-C), n = 5 to 10. Boxplots display the median, interquartile range (IQR), with the identification of the furthest values from the median not exceeding 1.5 × IQR. Statistical significance was determined by Shapiro-Wilk, then (A) Kruskal-Wallis and one-sided Wilcoxon rank-sum hypothesizing vaccine-associated induction compared to vehicle control, and two-sided Wilcoxon rank-sum test comparing age groups, (B) two-sided Wilcoxon rank-sum, (C) two-sided T-test, with significance annotated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
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