Enhanced durability of a Zika virus self-amplifying RNA vaccine through combinatorial OX40 and 4-1BB agonism
Hsueh-Han Lu, Rúbens Prince dos Santos Alves, Qin Hui Li, Luke Eder, Julia Timis, Henry Madany, Kantinan Chuensirikulchai, Krithik V. Varghese, Aditi Singh, Linda Le Tran, Audrey Street, Annie Elong Ngono, Michael Croft, Sujan Shresta
Hsueh-Han Lu, Rúbens Prince dos Santos Alves, Qin Hui Li, Luke Eder, Julia Timis, Henry Madany, Kantinan Chuensirikulchai, Krithik V. Varghese, Aditi Singh, Linda Le Tran, Audrey Street, Annie Elong Ngono, Michael Croft, Sujan Shresta
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Research Article Immunology Infectious disease Vaccines

Enhanced durability of a Zika virus self-amplifying RNA vaccine through combinatorial OX40 and 4-1BB agonism

Abstract

The SARS-CoV-2 pandemic highlighted the potential of mRNA vaccines in rapidly responding to emerging pathogens. However, immunity induced by conventional mRNA vaccines wanes quickly, requiring frequent boosters. Self-amplifying RNA (saRNA) vaccines, which extend antigen expression via self-replication, offer a promising strategy to induce more durable immune responses. In this study, we developed an saRNA vaccine encoding Zika virus (ZIKV) membrane and envelope proteins and evaluated its efficacy in mice. A single vaccination elicited strong humoral and cellular immune responses and reduced viral loads but only for 28 days. By day 84, antibody titers and T cell responses had significantly declined, resulting in reduced efficacy. To address this, we evaluated agonist antibodies targeting the T cell costimulatory molecules OX40 and 4-1BB. Coadministration of agonist antibodies enhanced CD8+ T cell responses to vaccination, resulting in sustained immunity and reduced viral loads at day 84. Depletion and passive transfer studies verified that long-term antiviral immunity was primarily CD8+ T cell dependent, with minimal contributions from antibody responses. These findings suggest that agonists targeting members of the tumor necrosis receptor superfamily, such as OX40 and 4-1BB, might enhance the durability of saRNA vaccine–induced protection, addressing a key limitation of current mRNA vaccine platforms.

Authors

Hsueh-Han Lu, Rúbens Prince dos Santos Alves, Qin Hui Li, Luke Eder, Julia Timis, Henry Madany, Kantinan Chuensirikulchai, Krithik V. Varghese, Aditi Singh, Linda Le Tran, Audrey Street, Annie Elong Ngono, Michael Croft, Sujan Shresta

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Figure 2

Immunogenicity of ZIKV M/E saRNA vaccine.

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Immunogenicity of ZIKV M/E saRNA vaccine. (A) Experimental protocol: Wil...
(A) Experimental protocol: Wild-type (WT) mice were immunized twice (days 0 and 28) with ZIKV M/E or Rluc vaccine (5 μg, intramuscularly) and bled on day 42. Sera were prepared, pooled, and injected intraperitoneally (400 μL/mouse) into naive Ifnar1−/− mice, which were retro-orbitally challenged 1 day later with 103 FFU of ZIKV SD001. Blood and organs were harvested on day 3 postinfection, and viral loads were analyzed by FFA. (B and C) Quantification of anti-ZIKV E IgG titers (B) and neutralizing antibody titers (C). (D) Quantification of ZIKV infectious particles in the indicated tissues. (E) Experimental protocol: WT mice were immunized twice (days 0 and 28) as above, and spleens were harvested on day 42. Splenocytes were stimulated in vitro with the indicated ZIKV peptides and then stained and analyzed by flow cytometry. (F and G) Number of IFN-γ+–producing, polyfunctional (IFN-γ+TNF-α+ or IFN-γ+TNF-α+IL-2+), and cytotoxic (IFN-γ+CD107a+) CD4+ (F) and CD8+ (G) effector memory T cells (CD3+CD4+CD44+CD62L−) (see Supplemental Figure 7 for gating strategy). Data are pooled from 2 independent experiments and are presented as the mean ± SEM. In panel D, for Ifnar1−/− mice, n = 7 for the Rluc group, and n = 8 for the ZIKV M/E group. In panels B, C, F, and G, for WT mice, n = 10 per group. Circles represent individual mice. Dotted line indicates the limit of detection. ***P < 0.001 by the Mann-Whitney test (B and C). *P < 0.05, ***P < 0.001 by the unpaired t test (D, F, and G).