CD137L adjuvanted vaccine increases antigen-specific T-cell responses but impacts antigen-specific humoral responses

CD137L adjuvanted vaccine increases antigen-specific T-cell responses but impacts antigen-specific humoral responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report CD137L adjuvanted vaccine increases antigen-specific T-cell responses but impacts antigen-specific humoral responses Kanin Salao, Ming Xuan Lim, Daryl Zhang Wei Lee, Jaz Linn Ng, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9365361/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract CD137 engagement through CD137 ligand (CD137L) or agonistic antibodies has shown promising results in cancer immunotherapy. Here, we explored CD137L as an adjuvant to improve vaccine-induced T-cell mediated immunity. Here we show that including CD137L in cis in mRNA vaccine significantly boosted T-cell responses, but not in protein vaccine. Furthermore, CD137L-containing vaccines induced lower antibody responses. These findings support that CD137L may boost the efficacy of T-cell based mRNA vaccines. Biological sciences/Cancer Biological sciences/Immunology Figures Figure 1 Figure 2 Figure 3 Main text T-cell responses are often neglected when evaluating the immunogenicity of vaccine candidates designed to confer antibody-mediated protection. However, recent research has shown that pre-existing cross-reactive T-cells can offer protection against severe COVID-19 disease ( 1 , 2 ), or influenza infections ( 3 – 6 ) and that vaccine-induced T-cell responses are emerging as good correlates of protection against symptomatic disease, including in children ( 7 – 9 ). In particular, robust cross-protective T-cell responses are important especially in the context of pathogens with rapid antigenic drift ( 10 ) or rapid antibody waning ( 11 ). Hence, strategies to improve the strength and breadth of T-cell responses upon vaccination are urgently needed. CD137 is a potent costimulatory molecule that is expressed on activated T-cells and NK cells ( 12 ). These effector cells receive co-stimulation when they interact with cognate CD137 ligand (CD137L) on antigen presenting cells (APC). CD137L also named 4-1BBL or TNFSF9 is a member of the TNF ligand superfamily. Signalling through CD137 is very potent and facilitates the elimination of malignancies in a large number of preclinical models which formed the basis for the development of agonistic anti-CD137 antibodies for tumor immunotherapy ( 13 ). Recently, Bowen et al. , attempted to capitalize on the Th1-adjuvanting properties of CD137L to improve the efficacy of a lead subunit vaccine candidate against Yersinia pestis ( 14 ). The reformulated subunit vaccine included a non-toxic soluble form of CD137L (SA-4-1BBL) which generated a robust CD4 T helper 1 (Th1) cellular and humoral responses in mice in the context of a Th2 polarizing adjuvant. Therefore, we hypothesized that including CD137L as a co-stimulator during vaccination with viral antigen vaccine candidates could potentiate known protective humoral and Th1 cellular responses and potentially delay antibody waning. We designed an mRNA vaccine construct that expresses M2 and NP vaccine antigen candidates of influenza A (strain H1N1/PR8) with or without co-expression of murine CD137L (mCD137L, Fig. 1 A). To investigate the antigen-specific T-cell responses, splenocytes were re-stimulated with 23-amino acid M2e peptide for CD4 or with immunodominant CD8 peptide NP 366–374 ( 15 ). The results show increased CD4 and CD8 T-cell responses in the mCD137L vaccinated group at 2 weeks post boost (Fig. 1 B-C), which persisted at 3 months post boost for the CD8 T-cell responses (Fig. 1 D-E). In particular we observed increased percentages of CD8 T-cells positive for IFNg and degranulation marker CD107a ( 16 ), supporting the induction of persistent CD8 T-cell killing function. We next characterized the M2e- and NP-specific antibody responses. Mice vaccinated with CD137L-M2-NP mRNA displayed significantly lower anti-M2e IgG titres both at pre- and post-boost compared to control mice immunized with M2-NP mRNA (Fig. 2 A). This was accompanied by a reduction in the percentage of M2e-specific germinal centre (GC) B-cells in the spleen (Fig. 2 B). Interestingly, the anti-NP antibody titres were comparable between both immunized groups (Fig. 2 C), suggesting that the influence of mCD137L on antibody production is antigen dependent. A longitudinal study up to 3 months post-boost indicates that while mCD137L influenced the magnitude of the M2-specific (Fig. 2 D) but not the NP-specific antibody response, it did not influence the durability of these antibody responses (Fig. 2 E). Together these results indicate that including mCD137L in cis in M2-NP mRNA vaccine construct improved the magnitude of antigen-specific T-cell responses in mice, including persistent functional CD8 T-cell responses, but significantly impaired the anti-M2e humoral responses. However, it did not affect the anti-NP antibody responses. We next assessed the effects of mCD137L on the cellular and humoral immune responses upon immunization with a recombinant protein-based vaccine. M2 protein is a transmembrane tetramer difficult to express and purify in its native form, while its 23-aminoacid peptide extracellular domain M2e is too poorly immunogenic. Therefore, we selected SARS-CoV2 RBD as protein-based vaccine prototype. Mice were immunized with recombinant SARS-CoV-2 RBD protein co-administered or not (control) with soluble extracellular domain of mCD137L. The protein suspensions were adjuvanted with Addavax™ a squalene-based oil-in-water nanoemulsion and a mimetic of MF59® that has been approved for human use ( 17 ). The RBD-specific T-cell responses were assessed upon re-stimulation of splenocytes with an overlapping RBD peptide pool. A trend towards increased numbers of CD4 T-cells secreting IL-4, IL-5 and IL-10 were observed in the mCD137L-treated group (Fig. 3 A), suggestive of a stronger Th2 polarized RBD-specific response, while the number of follicular helper T-cells (TFH CD4 + PD1 + CXCR5 + CD69 +/− and TFH AIM + ) and tissue resident CD4 T-cells (TRM CD4 + CD69 + CD103 + ) were comparable between both immunized groups (Fig. 3 A). Furthermore, and contrary to the mRNA-LNP format, there were no significant differences between both vaccinated groups in the number of cytokine-secreting RBD-specific CD8 T-cells (Tc1, Tc2, Tc17 and CD8 + GZMB + ) while the number of CD8 T-cells with tissue-resident markers (TRM CD8 + CD69 + CD103 + ) was increased in the mCD137L group (Fig. 3 B). Similar to the RNA-LNP format, we observed lower anti-RBD total IgG titers and neutralizing antibody responses in the mCD137L group (Fig. 3 C). However, and contrary to M2-NP mRNA immunization, comparable percentages of GC, memory and plasma B-cells (total and antigen-specific B cells) were measured between both immunized groups (Fig. 3 D). A lower trend in the percentage of plasmablasts was nevertheless noted in the mCD137L group (Fig. 3 D). Together, our data supports that adjuvanting a protein-based vaccine with CD137L does not significantly improve the antigen-specific T-cell response but reduces the antibody response. The absence of a clear effect of CD137L on the T-cell responses may be attributed to the presence of Addavax™. Indeed, Addavax™ is a strong inducer of a broad range of cytokines, potentiating helper type 1 and follicular helper T-cell formation in the germinal centres ( 18 ). Therefore, it is possible that any CD137L effect may be masked by Addavax™. Of note, in this recombinant protein format, CD137L is provided in trans and Otano et al. have previously shown that CD8 T-cell co-stimulation via CD137-CD137L is significantly more potent in cis than in trans ( 19 ). In contrast, the suppressive effect of mCD137L on the antibody responses seems independent of the vaccine format ( cis versus trans ). Previous studies have reported the effects of CD137L-CD137 interaction on antibody titres and B-cells alongside T-cell parameters. CD137L-deficient mice developed normal humoral responses against vesicular stomatitis virus (VSV), decreased CTL responses to lipidated lymphocytic choriomeningitis virus (LCMV) peptide immunization, and decreased CTL responses with little effect on CD4 T-cell and B-cell responses during LCMV infection ( 20 – 22 ). A subsequent study confirmed that CD137 KO mice (CD137 −/− ) had similar anti-VSV antibody levels and lower CTL responses after infection compared to wildtype counterparts ( 23 ). However, contrary to earlier reports, the same study found decreased anti-keyhole limpet hemocyanin (KLH) IgG2a and IgG3 after KLH immunization, and altered functionality of both CD4 and CD8 T-cell subsets ( 23 ). During severe influenza infection, CD137L −/− mice showed decreased CD8 T-cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality, which could be rescued by low intranasal dose of adenoviral CD137L delivery; whereas high dose delivery worsened the infection outcome, although the antibody titres were not assessed ( 24 ). The lower antibody responses we observed against M2e and RBD may result from the competition between vaccine-produced mCD137L and endogenous mCD137L expressed on antigen specific B-cells, potentially minimizing interactions between B and T-cells via the CD137-CD137L axis, thereby preventing full B cell activation. Consistent with this, B-cell proliferation and antibody synthesis were increased by 5- and 3-fold, respectively, when cultured on immobilized CD137 substrate ( 25 ). Of note, contrary to human B cells, murine B cells do not express CD137 ( 26 – 28 ), hence ruling out that vaccine-produced mCD137L directly interacts with B cells in vaccinated mice. A similar downregulating effect of CD137 stimulation on antibody levels has been previously described upon CD137 agonistic antibody administration during chikungunya virus infection ( 29 , 30 ), with a drastic effect on germinal centre composition and organisation. It is possible that the high dose of anti-CD137 antibody administered (400µg), prevented endogenous CD137L binding. Interestingly, in the latter study, while the authors reported a strong inhibitory effect of the CD137 agonist antibody on a T-cell dependent antigen (4-hydroxy-3-nitrophenylacetyl hapten conjugated with KLH), no effect on antibodies was observed against a T-cell independent antigen (4-hydroxy-3-nitrophenylacetyl hapten-ficoll complex) ( 30 ), further supporting the involvement of T-cells in mCD137L-mediated inhibition of B-cell activation. In our study however both M2e- and NP-specific antibody responses are expected to be T-cell dependent (31, 32), and the exact mechanism underlying the mCD137L-mediated suppression of M2e- but not NP-specific antibody responses remains unclear. The intracellular localization of NP implies that NP-specific B-cells interact with NP after NP producing cells died and released the antigen, perhaps minimizing interference with surface-expressed CD137L. In conclusion, our work supports that incorporating CD137L in cis within an mRNA–LNP platform may offer a useful way to tune vaccine immunity toward strong, sustained T-cell responses while limiting antibody production against a defined T-cell–dependent antigen. This vaccine strategy could be particularly advantageous against pathogens for which T-cells have been established as immune correlates of protection, and/or when antibody responses are undesirable, including infectious diseases for which antibody-dependent enhancement is an issue, as exemplified during Dengue vaccination or the early anti-RSV vaccines designs (reviewed in (33)). Materials and Methods Recombinant proteins and peptides Purified NP protein was purchased from Sino Biological (40776-V08B-100). M2e sequence used was MSLLTEVETPIRNEWGCRCNDSSD and was custom made by Genscript. NP 366–374 peptide (TYQRTRALV) was purchased from Genscript. Alpha pool of 53 overlapping peptides from SARS-CoV-2 RBD B.1.1.7 was purchased from Genscript (RP30026). Peptide pool was reconstituted in DMSO (D2650, Sigma-Aldrich). Purified SARS CoV 2 MBP-RBD and MBP-mCD137L recombinant proteins were produced by the Antibody Core Facility, LSI, NUS. Briefly, genes encoding SARS-CoV-2-RBD (aa 319–591 of SARS-CoV-2 spike QVX50359.1) or murine CD137L (aa 104–309 of NP_033430.1) were cloned into expression vector pHLmMBP-10 (Addgene) allowing expression of fusion proteins with N-terminal MBP and hexahistidine tags. MBP-RBD and MBP-mCD137L proteins were expressed in Expi293F cells (ThermoFisher Scientific, RRID: CVCL_D615) using Expi293 System. Culture supernatants were harvested on day 5 post transfection, and supernatants were incubated with nickel beads (Roche) at 4°C for 1 h. Nickel beads were equilibrated with binding buffer (25 mM Tris, 300 mM NaCl, pH 8.0) prior to incubation with culture supernatants. Supernatants containing beads were passed through cOmplete His-Tag Purification column (Roche) twice, and column was washed with binding buffer containing increasing concentrations of Imidazole. Proteins were eluted with elution buffer (25 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0), concentrated down and buffer-exchanged into 25 mM Tris, 300 mM NaCl, pH 8.0, and quantified by NanoDrop A280. Purity was assessed by standard denaturing polyacrylamide gel electrophoresis. Mouse immunisation For Flu vaccination model, 5–6 week old female Balb/c mice (InVivos) were injected intramuscularly (i.m.) in the hind leg with 5 µg of M2-NP mRNA-LNP or 7.5 µg of CD137L-M2-NP mRNA-LNP in a total volume of 50 µl (in PBS) in a prime-boost regimen with 3-week interval. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of NUS under protocol number R23-0588. For SARS-CoV-2 immunization model, 6–8 week old C57BL/6 mice male or female (single sex per experimental replicate) were injected twice at 3-week interval i.m. in the hind leg with 20 µg SARS CoV 2 MBP-RBD and 25 µL of AddaVax™ adjuvant (vac-adx-10, InvivoGen) with or without 20 µg of mCD137L (in a final volume of 50 µL in PBS). All mice experiments were performed under IACUC protocol 231806 and in accordance to the National Advisory NACLAR guidelines. Serum collection Blood was collected under light anaesthesia (isoflurane 4% for induction, 1–2% for maintenance. Blood was left to clot for 1 h at room temperature, centrifuged at 2,500 g for 10 min at 10°C to acquire serum. Serum was then heat inactivated at 56°C for 30 min and stored at -80°C before downstream ELISA. Preparation of splenocyte suspensions Spleens were harvested from euthanized mice and single cell suspensions were prepared by mechanical disruption and filtration through 40mm cell strainer in complete RPMI (cRPMI) (RPMI 1640 medium + 10% FBS + 1% Pen/Strep). Cell suspensions were centrifuged at 400 xg for 5 min at 10°C, red blood cells were lysed with Flow Cytometry Mouse Lyse Buffer (FC003, R&D Systems) at room temperature for 5 min. Cell suspensions were then quenched with cRPMI, centrifuged again at 400 xg for 5 min at 10°C and resuspended with cRPMI Flow cytometry For M2e-and NP specific T-cell responses, freshly prepared splenocyte suspensions (2x10 6 ) were seeded into U-bottom 96-well plates. A final concentration of 20 µg/mL of NP 366–374 peptide or M2e peptide was added into each relevant well and the plate was incubated together with anti-CD107a antibody (BioLegend) (1:100 dilution) for 16 h at 37°C, 5% CO 2 . Next, brefeldin A (1:100 dilution) (ThermoFisher Scientific) was added and incubated further for 3 h. For the positive control, a final concentration of 50 ng/mL Phorbol 12-myristate 13-acetate (PMA) (Invivogen,) and 1µg/mL of ionomycin (Invivogen) were added. For the negative control, 0.25% DMSO in cRPMI was added. The plate was incubated for 4 h at 37°C, 5% CO2 before the cells were washed with 1xPBS. The cells were stained with Near IR live-dead dye (1000x dilution) (ThermoFisher Scientific) for 15 min in the dark at 4°C. Next, the cells were incubated with FcR blocker (25x dilution) (Miltenyi Biotec) for 30 min at 4°C. For both panels, the cells were washed with FACs buffer before staining of cell surface markers (Table S1 ). The cells were resuspended in fix/perm buffer (ThermoFisher Scientific) and incubated for 30 min in the dark at 4°C. The cells were then washed with permeabilization buffer (ThermoFisher Scientific) and stained for intracellular cytokines (Table S1 ) for 30 min in the dark at 4°C. Final washes with permeabilization and then FACs buffer were done before flow cytometry analysis. For RBD specific T-cells, freshly prepared splenocyte suspensions (2x10 6 ) were stimulated overnight at 37°C with SARS-CoV-2 RBD peptide pool at 2.5 µg/mL with mouse IL-2 at 30U/mL (212-12-20UG, PeproTech) in complete IMDM (cIMDM) (IMDM + 10% FBS + 1% Pen/Strep). Brefeldin A (420601, Biolegend) and Monensin (420701, Biolegend) diluted in cIMDM were next added and incubated at 37°C for 3 h. Splenocytes were then stained with Live-Dead Fixable Blue (L23105, Invitrogen) for 5 min at 4°C in the dark, and blocked with TruStain FcX™ PLUS (anti-mouse CD16/32) (156604, Biolegend) in MACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) for 15 min. Splenocytes were then stained for extracellular T-cell markers (Table S2 ) for 30 min at 4°C. Streptavidin-StarBright Violet (STAR210SBV515, Biorad) was added for B cell, Macrophage and NK cell exclusion gate. Cells were fixed with BD FACS™ Lysing Solution (349202, BD) and then permeabilised with BD FACS™ Permeabilizing Solution (340973, BD). After permeabilisation, splenocytes were blocked again before staining for intracellular T-cell markers (Table S2 ) at 4°C overnight. For M2e specific GC B cells, M2e-specific GC B cells were detected using biotinylated M2e in combination with fluorophore-conjugated SAv. Biotinylated M2e was multimerized at a 4:1 molar ratio with SAv-BV421 and 24 SAv-PE (BD Biosciences) respectively. SAv-FITC was used as decoy probe without biotinylated M2e to exclude cells with non-specific binding to SAv. After multimerization, M2e probe master mix was made by mixing the fluorophore-conjugated M2e probes with 5 µM free D-biotin (Thermo Fisher Scientific, B20656) to minimize cross-reactivity between probes, diluted in a 1:3 mixture of Brilliant Stain buffer (BD Biosciences) and FACS buffer (2% FBS + 1 mM EDTA in PBS). Cells were then incubated with anti-CD16/CD32 Fc block (BD Biosciences) and eFluor 780 Fixable Viability Dye (Thermo Fisher Scientific) diluted in FACS buffer (1:200 and 1:1,000 respectively) for 20 min at 4°C. Cells were washed with FACS buffer and stained with respective decoy probe (Table S3) diluted in FACS buffer with 5 µM free D-biotin for 30 min at 4°C in the dark. Subsequently, cells were washed and stained with M2e probe master mix for 1h at 4°C in the dark. Thereafter, cells were washed and stained with GC B cell surface marker antibodies (Table S3) diluted in Brilliant Stain and FACS buffer for 30 min at 4°C in the dark. For RBD specific B cells, freshly prepared splenocytes (5x10 6 ) were stained with Live-Dead Fixable Blue (L23105, Invitrogen) for 10 min at 4°C in the dark, then blocked with TruStain FcX™ PLUS (anti-mouse CD16/32) (156604, Biolegend) in MACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) for 15 min. RBD specific B cell subsets were detected by using biotinylated SARS-CoV-2 RBD protein (793906, Biolegend) multimerised with Streptavidin-PE-Fire 700 (405174, Biolegend) at molar ratio of 4:1 in a 50/50 mixture of PBS with 2% FBS and MACS buffer as previously described ( 31 ). After multimerization Streptavidin-StarBright Violet (STAR210SBV515, Biorad) was added as a decoy to gate ouT-cells that bind non-specifically to RBD. Then 5 µM free D-biotin (B20656; Thermo Fisher Scientific) was added to minimise probe cross reactivity. Cells were then stained with RBD probe mastermix for 1h at 4°C in the dark. After incubation, cells were then stained with the B cell markers listed in (Table S4) for 30 min at 4°C. Cells were then fixed with BD FACS™ Lysing Solution (349202, BD). Enzyme-Linked Immunosorbent Assay (ELISA) For anti-M2e-specific antibody titres, 96-well plates were coated with M2e (150ng per well) and incubated overnight at 4°C. The plate was then washed three times with wash buffer (1x PBS, 0.05% Tween-20), then blocked with blocking buffer (1x PBS, 1% Bovine Serum Albumin for 1 h at 37°C. The plate was then washed once with wash buffer before adding serially diluted (two-fold) mice sera and incubated at 37°C for 1 h. The plate was washed thrice before the addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (Bio-Rad, 170–6516) at 1:3,000 (in 1% BSA). After incubation at 37°C for 1 h, the plates were washed three times. The o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich, P9187-50SET) was added to each well and incubated at room temperature for 10 min in the dark. H 2 SO 4 was added to each well to stop the reaction, and absorbance was read at 490 nm. Antibody titres were determined by non-linear regression as the reciprocal of the highest serum dilution with absorbance corresponding to three times the absorbance of blank wells. For anti-NP IgG, 96-well plates were coated with NP (150ng per well) and incubated overnight at 4°C. The plate was then washed three times with wash buffer (1x PBS, 0.05% Tween-20), then blocked with blocking buffer (1x PBS, 1% Bovine Serum Albumin for 1 h at 37°C. The plate was then washed once with wash buffer before adding serially diluted (two-fold) mice sera and incubated at 37°C for 1 h. The plate was then washed thrice before the addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (Bio-Rad, 170–6516) at 1:3,000 (in 1% BSA). After incubation at 37°C for 1 h, the plates were then washed three times. The o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich, P9187-50SET) was added to each well and incubated at room temperature for 10 min in the dark. H 2 SO 4 was added to each well to stop the reaction, and absorbance was read at 490 nm. Antibody titres were determined by non-linear regression as the reciprocal of the highest serum dilution with absorbance corresponding to three times the absorbance of blank wells. For anti-RBD IgG, MaxiSorp Immuno plates (442404, Thermo Scientific) were coated with purified SARS CoV 2 MBP-RBD at 10 µg/mL and incubated overnight at 4°C. Mouse sera were diluted 1:500 in antibody dilution buffer [1X PBS, 0.5% Bovine Serum Albumin (BSA) (SLCQ7756, Sigma-Aldrich), 0.05% Tween20 (P1379, Sigma-Aldrich)]. Plates were washed twice with washing buffer (1X PBS, 0.05% Tween20) before addition of diluted mouse sera (1:500 dilution) and incubated at 37°C for 1 h. Plates were then washed twice with washing buffer before addition of Goat anti-mouse IgG (H + L) Secondary antibody HRP (1:1000) (31430, Invitrogen) diluted in antibody dilution buffer and incubated at 37°C for 30 min. After incubation, the plates were washed twice with washing buffer and once with distilled water. TMB substrate (T8665, Sigma-Aldrich) was added, and plates were incubated in the dark for 6 min, reaction was stopped with 0.16 M H 2 SO 4 (S5814, Sigma-Aldrich). Absorbance was read at 450 nm (reference 690nm) on a Tecan plate reader. Surrogate Virus Neutralization Test cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (L00847-A, Genscript) was used to detect SARS-CoV-2 neutralizing antibodies in mouse sera (1:10 dilution) as per the manufacturer’s protocol. Data acquisition and analysis Stained splenocytes were acquired on Novocyte Penteon (Agilent Technologies) and data analysis was performed on FlowJo (v.10.9.0). Gating strategies for each antibody panel are shown in Figure S1 . Figures and statistics were plotted using GraphPad Prism (v10.5.0). Declarations Competing Interests The authors declare no competing interests. Author Contribution KS, MXL, DZWL, JLN, WL and EG performed the experiments.J.L.L. and E.N. produced the recombinant proteins.KS, HS, SA and GC designed the experiments, analysed the data and wrote the manuscript. Acknowledgments The study was funded by the Programme for Research in Epidemic Preparedness And REsponse (PREPARE) awarded to HS (PREPARE-OC-VT-2022-008). Part of this work was also supported by A*STAR core funding and NMRC OF-IRG (MOH-001618-00) awarded to GC. We thank A*STAR IDL Pathogen Flow platform team, as well as A/P Paul Hutchinson and his team from the flow cytometry core facility at Life Sciences Institute, NUS for their invaluable help in this study. 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CD137 (4-1BB) costimulation of CD8(+) T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation. Nat Commun 12:7296. DeBenedette MA, Wen T, Bachmann MF, Ohashi PS, Barber BH, Stocking KL, Peschon JJ, Watts TH. 1999. Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J Immunol 163:4833–41. Tan JT, Whitmire JK, Murali-Krishna K, Ahmed R, Altman JD, Mittler RS, Sette A, Pearson TC, Larsen CP. 2000. 4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination. J Immunol 164:2320–5. Tan JT, Whitmire JK, Ahmed R, Pearson TC, Larsen CP. 1999. 4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses. J Immunol 163:4859–68. Kwon BS, Hurtado JC, Lee ZH, Kwack KB, Seo SK, Choi BK, Koller BH, Wolisi G, Broxmeyer HE, Vinay DS. 2002. Immune responses in 4-1BB (CD137)-deficient mice. J Immunol 168:5483–90. Lin GH, Sedgmen BJ, Moraes TJ, Snell LM, Topham DJ, Watts TH. 2009. Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease. J Immunol 182:934–47. Pauly S, Broll K, Wittmann M, Giegerich G, Schwarz H. 2002. CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers. J Leukoc Biol 72:35–42. Pollok KE, Kim YJ, Hurtado J, Zhou Z, Kim KK, Kwon BS. 1994. 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur J Immunol 24:367–74. Zhang X, Voskens CJ, Sallin M, Maniar A, Montes CL, Zhang Y, Lin W, Li G, Burch E, Tan M, Hertzano R, Chapoval AI, Tamada K, Gastman BR, Schulze DH, Strome SE. 2010. CD137 promotes proliferation and survival of human B cells. J Immunol 184:787–95. Futagawa T, Akiba H, Kodama T, Takeda K, Hosoda Y, Yagita H, Okumura K. 2002. Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int Immunol 14:275–86. Hong JP, McCarthy MK, Davenport BJ, Morrison TE, Diamond MS. 2019. Clearance of Chikungunya Virus Infection in Lymphoid Tissues Is Promoted by Treatment with an Agonistic Anti-CD137 Antibody. J Virol 93. Hong JP, Reynoso GV, Andhey PS, Swain A, Turner JS, Boon ACM, Krammer F, Ellebedy AH, Zanini F, Artyomov M, Hickman HD, Diamond MS. 2020. An Agonistic Anti-CD137 Antibody Disrupts Lymphoid Follicle Structure and T-Cell-Dependent Antibody Responses. Cell Rep Med 1. Cheang NYZ, Tan KS, Tan PS, Purushotorma K, Yap WC, Tullett KM, Chua BYL, Yeoh AY, Tan CQH, Qian X, Chen H, Tay DJW, Caminschi I, Tan YJ, Macary PA, Tan CW, Lahoud MH, Alonso S. 2024. Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice. Mol Ther 32:2299–2315. Additional Declarations No competing interests reported. Supplementary Files Salaoetal2026supplementalmaterial.docx Salaoetal2026SupplementalFile1.xlsx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 12 May, 2026 Reviews received at journal 04 May, 2026 Reviews received at journal 04 May, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Submission checks completed at journal 12 Apr, 2026 First submitted to journal 09 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9365361","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":627518180,"identity":"99c4455a-7003-4ada-b51b-3e4fb75df887","order_by":0,"name":"Kanin Salao","email":"","orcid":"","institution":"Immunology Translational Research Programme, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Kanin","middleName":"","lastName":"Salao","suffix":""},{"id":627518182,"identity":"1745c998-69d0-438b-9da7-957c0ae5eb99","order_by":1,"name":"Ming Xuan Lim","email":"","orcid":"","institution":"A*STAR Infectious Diseases Labs (A*STAR IDL), Agency for Science, Technology and Research (A*STAR)","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"Xuan","lastName":"Lim","suffix":""},{"id":627518187,"identity":"27ee1966-8a46-4c3c-95eb-1321fa1391df","order_by":2,"name":"Daryl Zhang Wei Lee","email":"","orcid":"","institution":"Immunology Programme, Life Sciences Institute, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Daryl","middleName":"Zhang Wei","lastName":"Lee","suffix":""},{"id":627518191,"identity":"382f7b97-91bc-4571-adae-d875c258dd4e","order_by":3,"name":"Jaz Linn Ng","email":"","orcid":"","institution":"Immunology Programme, Life Sciences Institute, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Jaz","middleName":"Linn","lastName":"Ng","suffix":""},{"id":627518195,"identity":"5d04aa41-b84e-492d-8d71-e4e935296172","order_by":4,"name":"Jia Le Lin","email":"","orcid":"","institution":"Immunology Translational Research Programme, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"Le","lastName":"Lin","suffix":""},{"id":627518198,"identity":"aa44a9ae-b956-4127-a191-160658ec4454","order_by":5,"name":"Wanzhen Lu","email":"","orcid":"","institution":"A*STAR Infectious Diseases Labs (A*STAR IDL), Agency for Science, Technology and Research (A*STAR)","correspondingAuthor":false,"prefix":"","firstName":"Wanzhen","middleName":"","lastName":"Lu","suffix":""},{"id":627518200,"identity":"283b029a-7ba5-4838-aac0-9621b14b2346","order_by":6,"name":"Estelle Goh","email":"","orcid":"","institution":"A*STAR Infectious Diseases Labs (A*STAR IDL), Agency for Science, Technology and Research (A*STAR)","correspondingAuthor":false,"prefix":"","firstName":"Estelle","middleName":"","lastName":"Goh","suffix":""},{"id":627518203,"identity":"dc09621d-40f8-4edc-9d1d-a72f2833ebbb","order_by":7,"name":"Emily Nickles","email":"","orcid":"","institution":"Immunology Translational Research Programme, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Nickles","suffix":""},{"id":627518207,"identity":"2563139e-a163-4326-8f4e-00921d1724b8","order_by":8,"name":"Herbert Schwarz","email":"","orcid":"","institution":"Immunology Translational Research Programme, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Herbert","middleName":"","lastName":"Schwarz","suffix":""},{"id":627518210,"identity":"0aee87ea-8b19-4bb0-863b-ad61cbd8e0ed","order_by":9,"name":"Sylvie Alonso","email":"","orcid":"","institution":"Infectious Diseases Translational Research Programme; Department of Microbiology \u0026 Immunology; Yong Loo Lin School of Medicine, National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Sylvie","middleName":"","lastName":"Alonso","suffix":""},{"id":627518213,"identity":"61b919fa-67c5-4967-87b9-2b8d921ed4bb","order_by":10,"name":"Guillaume Carissimo","email":"data:image/png;base64,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","orcid":"","institution":"A*STAR Infectious Diseases Labs (A*STAR IDL), Agency for Science, Technology and Research (A*STAR)","correspondingAuthor":true,"prefix":"","firstName":"Guillaume","middleName":"","lastName":"Carissimo","suffix":""}],"badges":[],"createdAt":"2026-04-09 08:23:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9365361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9365361/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107868870,"identity":"f93a74f7-1a1e-4dd3-b0df-cb91d76d57d9","added_by":"auto","created_at":"2026-04-27 07:34:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16732429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT-cell responses in CD137L-M2-NP mRNA immunized mice.\u003c/strong\u003e (A) Design of the vaccine constructs and predicted subcellular localization after expression. Adult female BALB/c mice were immunized intramuscularly (i.m.) twice at a 3-week interval with either 5 μg of M2-NP mRNA -LNP or 7.5 μg of CD137L-M2-NP mRNA-LNP. (B–C) Two weeks after the booster immunization, splenocytes were restimulated with (B) M2e peptide, followed by intracellular staining for TNF-α, IFN-γ, and IL-2, or with (C) NP\u003csub\u003e366–374 \u003c/sub\u003epeptide to assess CD8⁺ T-cell responses. (D–E) Three months after the booster immunization, splenocytes were restimulated with (D) M2e peptide or (E) NP\u003csub\u003e366–374 \u003c/sub\u003epeptide and analysed for functionality of CD4 and CD8 T-cells respectively. Data are presented as the percentage of the parent population as indicated. (n=5-10 per group)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9365361/v1/e96205b4ba9b4737b2686f74.png"},{"id":107868863,"identity":"a06a0cf7-9eca-4db4-9ed9-b8ebf2459389","added_by":"auto","created_at":"2026-04-27 07:34:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15255063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody responses in CD137L-M2-NP mRNA immunized mice.\u003c/strong\u003e Adult female BALB/c mice were immunized as indicated under Figure 1. (A) Anti-M2e IgG titers were measured by ELISA in serum harvested at1 week before and 2 weeks after the boost. (B) M2e-specific germinal center (GC) B cells were analysed by flow cytometry in the spleen from immunized mice harvested at two weeks after the boost. (C) Anti-NP IgG titers were measured by ELISA in serum harvested at 1 week before and 2 weeks after the boost. M2e-specific (D) and NP-specific (E) IgG titers were measured longitudinally up to 3 months post-boost. (n=5-10 per\u003cem\u003e \u003c/em\u003egroup)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9365361/v1/4894087cafc08a5d52f86ae6.png"},{"id":107731869,"identity":"436823ac-0df5-481d-b572-d9d9bfd95109","added_by":"auto","created_at":"2026-04-24 13:12:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5811890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune responses in mice immunized with purified RBD and sCD137L proteins.\u003c/strong\u003e Adult female or males C57BL6J mice were vaccinated i.m. in a prime boost regimen at a 3-week interval with RBD (20 mg) alone or together with soluble CD137L (20 mg) formulated in Addavax\u003csup\u003eTM\u003c/sup\u003e. 2 weeks post boost, splenocytes were analysed by flow cytomerty or stimulated with an RBD peptide pool to assess functionality (A) CD4 T-cells, (B) CD8 T-cells.. (C) The anti-RBD IgG response was monitored over time post-boost by ELISA at a single dilution (1:500). The neutralizing activity was assessed by surrogate neutralization assay c-Pass\u003csup\u003eTM\u003c/sup\u003e 1:10 diluted individual sera (n=5-10/group). (D) Splenocytes were analysed by flow cytometry without stimulation for GC B-cells. (n=10-15 per group)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9365361/v1/72c05a8070034778e1f0aa11.png"},{"id":107731866,"identity":"30ee08db-8832-4c66-969b-f0cd41c4dc34","added_by":"auto","created_at":"2026-04-24 13:12:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4274525,"visible":true,"origin":"","legend":"","description":"","filename":"Salaoetal2026supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9365361/v1/0a029868918275e2cb80356e.docx"},{"id":107869033,"identity":"b04ef328-e064-48b1-9fc1-496777c326c0","added_by":"auto","created_at":"2026-04-27 07:35:49","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29314,"visible":true,"origin":"","legend":"","description":"","filename":"Salaoetal2026SupplementalFile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9365361/v1/a1162649ab8950bd91ecd19b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CD137L adjuvanted vaccine increases antigen-specific T-cell responses but impacts antigen-specific humoral responses","fulltext":[{"header":"Main text","content":"\u003cp\u003eT-cell responses are often neglected when evaluating the immunogenicity of vaccine candidates designed to confer antibody-mediated protection. However, recent research has shown that pre-existing cross-reactive T-cells can offer protection against severe COVID-19 disease (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), or influenza infections (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and that vaccine-induced T-cell responses are emerging as good correlates of protection against symptomatic disease, including in children (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In particular, robust cross-protective T-cell responses are important especially in the context of pathogens with rapid antigenic drift (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) or rapid antibody waning (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Hence, strategies to improve the strength and breadth of T-cell responses upon vaccination are urgently needed.\u003c/p\u003e \u003cp\u003eCD137 is a potent costimulatory molecule that is expressed on activated T-cells and NK cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). These effector cells receive co-stimulation when they interact with cognate CD137 ligand (CD137L) on antigen presenting cells (APC). CD137L also named 4-1BBL or TNFSF9 is a member of the TNF ligand superfamily. Signalling through CD137 is very potent and facilitates the elimination of malignancies in a large number of preclinical models which formed the basis for the development of agonistic anti-CD137 antibodies for tumor immunotherapy (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, Bowen \u003cem\u003eet al.\u003c/em\u003e, attempted to capitalize on the Th1-adjuvanting properties of CD137L to improve the efficacy of a lead subunit vaccine candidate against \u003cem\u003eYersinia pestis\u003c/em\u003e (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The reformulated subunit vaccine included a non-toxic soluble form of CD137L (SA-4-1BBL) which generated a robust CD4 T helper 1 (Th1) cellular and humoral responses in mice in the context of a Th2 polarizing adjuvant. Therefore, we hypothesized that including CD137L as a co-stimulator during vaccination with viral antigen vaccine candidates could potentiate known protective humoral and Th1 cellular responses and potentially delay antibody waning.\u003c/p\u003e \u003cp\u003eWe designed an mRNA vaccine construct that expresses M2 and NP vaccine antigen candidates of influenza A (strain H1N1/PR8) with or without co-expression of murine CD137L (mCD137L, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To investigate the antigen-specific T-cell responses, splenocytes were re-stimulated with 23-amino acid M2e peptide for CD4 or with immunodominant CD8 peptide NP\u003csub\u003e366\u0026ndash;374\u003c/sub\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The results show increased CD4 and CD8 T-cell responses in the mCD137L vaccinated group at 2 weeks post boost (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C), which persisted at 3 months post boost for the CD8 T-cell responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). In particular we observed increased percentages of CD8 T-cells positive for IFNg and degranulation marker CD107a (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), supporting the induction of persistent CD8 T-cell killing function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next characterized the M2e- and NP-specific antibody responses. Mice vaccinated with CD137L-M2-NP mRNA displayed significantly lower anti-M2e IgG titres both at pre- and post-boost compared to control mice immunized with M2-NP mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This was accompanied by a reduction in the percentage of M2e-specific germinal centre (GC) B-cells in the spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Interestingly, the anti-NP antibody titres were comparable between both immunized groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that the influence of mCD137L on antibody production is antigen dependent. A longitudinal study up to 3 months post-boost indicates that while mCD137L influenced the magnitude of the M2-specific (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) but not the NP-specific antibody response, it did not influence the durability of these antibody responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether these results indicate that including mCD137L in \u003cem\u003ecis\u003c/em\u003e in M2-NP mRNA vaccine construct improved the magnitude of antigen-specific T-cell responses in mice, including persistent functional CD8 T-cell responses, but significantly impaired the anti-M2e humoral responses. However, it did not affect the anti-NP antibody responses.\u003c/p\u003e \u003cp\u003eWe next assessed the effects of mCD137L on the cellular and humoral immune responses upon immunization with a recombinant protein-based vaccine. M2 protein is a transmembrane tetramer difficult to express and purify in its native form, while its 23-aminoacid peptide extracellular domain M2e is too poorly immunogenic. Therefore, we selected SARS-CoV2 RBD as protein-based vaccine prototype. Mice were immunized with recombinant SARS-CoV-2 RBD protein co-administered or not (control) with soluble extracellular domain of mCD137L. The protein suspensions were adjuvanted with Addavax\u0026trade; a squalene-based oil-in-water nanoemulsion and a mimetic of MF59\u0026reg; that has been approved for human use (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The RBD-specific T-cell responses were assessed upon re-stimulation of splenocytes with an overlapping RBD peptide pool. A trend towards increased numbers of CD4 T-cells secreting IL-4, IL-5 and IL-10 were observed in the mCD137L-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggestive of a stronger Th2 polarized RBD-specific response, while the number of follicular helper T-cells (TFH CD4\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003e+\u003c/sup\u003e CXCR5\u003csup\u003e+\u003c/sup\u003eCD69\u003csup\u003e+/\u0026minus;\u003c/sup\u003e and TFH AIM\u003csup\u003e+\u003c/sup\u003e) and tissue resident CD4 T-cells (TRM CD4\u003csup\u003e+\u003c/sup\u003eCD69\u003csup\u003e+\u003c/sup\u003eCD103\u003csup\u003e+\u003c/sup\u003e) were comparable between both immunized groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, and contrary to the mRNA-LNP format, there were no significant differences between both vaccinated groups in the number of cytokine-secreting RBD-specific CD8 T-cells (Tc1, Tc2, Tc17 and CD8\u003csup\u003e+\u003c/sup\u003eGZMB\u003csup\u003e+\u003c/sup\u003e) while the number of CD8 T-cells with tissue-resident markers (TRM CD8\u003csup\u003e+\u003c/sup\u003e CD69\u003csup\u003e+\u003c/sup\u003e CD103\u003csup\u003e+\u003c/sup\u003e) was increased in the mCD137L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar to the RNA-LNP format, we observed lower anti-RBD total IgG titers and neutralizing antibody responses in the mCD137L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, and contrary to M2-NP mRNA immunization, comparable percentages of GC, memory and plasma B-cells (total and antigen-specific B cells) were measured between both immunized groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). A lower trend in the percentage of plasmablasts was nevertheless noted in the mCD137L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTogether, our data supports that adjuvanting a protein-based vaccine with CD137L does not significantly improve the antigen-specific T-cell response but reduces the antibody response. The absence of a clear effect of CD137L on the T-cell responses may be attributed to the presence of Addavax\u0026trade;. Indeed, Addavax\u0026trade; is a strong inducer of a broad range of cytokines, potentiating helper type 1 and follicular helper T-cell formation in the germinal centres (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, it is possible that any CD137L effect may be masked by Addavax\u0026trade;. Of note, in this recombinant protein format, CD137L is provided in \u003cem\u003etrans\u003c/em\u003e and Otano \u003cem\u003eet al.\u003c/em\u003e have previously shown that CD8 T-cell co-stimulation via CD137-CD137L is significantly more potent in \u003cem\u003ecis\u003c/em\u003e than in \u003cem\u003etrans\u003c/em\u003e (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In contrast, the suppressive effect of mCD137L on the antibody responses seems independent of the vaccine format (\u003cem\u003ecis\u003c/em\u003e versus \u003cem\u003etrans\u003c/em\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have reported the effects of CD137L-CD137 interaction on antibody titres and B-cells alongside T-cell parameters. CD137L-deficient mice developed normal humoral responses against vesicular stomatitis virus (VSV), decreased CTL responses to lipidated lymphocytic choriomeningitis virus (LCMV) peptide immunization, and decreased CTL responses with little effect on CD4 T-cell and B-cell responses during LCMV infection (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). A subsequent study confirmed that CD137 KO mice (CD137\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) had similar anti-VSV antibody levels and lower CTL responses after infection compared to wildtype counterparts (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, contrary to earlier reports, the same study found decreased anti-keyhole limpet hemocyanin (KLH) IgG2a and IgG3 after KLH immunization, and altered functionality of both CD4 and CD8 T-cell subsets (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). During severe influenza infection, CD137L\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed decreased CD8 T-cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality, which could be rescued by low intranasal dose of adenoviral CD137L delivery; whereas high dose delivery worsened the infection outcome, although the antibody titres were not assessed (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The lower antibody responses we observed against M2e and RBD may result from the competition between vaccine-produced mCD137L and endogenous mCD137L expressed on antigen specific B-cells, potentially minimizing interactions between B and T-cells via the CD137-CD137L axis, thereby preventing full B cell activation. Consistent with this, B-cell proliferation and antibody synthesis were increased by 5- and 3-fold, respectively, when cultured on immobilized CD137 substrate (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Of note, contrary to human B cells, murine B cells do not express CD137 (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), hence ruling out that vaccine-produced mCD137L directly interacts with B cells in vaccinated mice.\u003c/p\u003e \u003cp\u003eA similar downregulating effect of CD137 stimulation on antibody levels has been previously described upon CD137 agonistic antibody administration during chikungunya virus infection (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), with a drastic effect on germinal centre composition and organisation. It is possible that the high dose of anti-CD137 antibody administered (400\u0026micro;g), prevented endogenous CD137L binding. Interestingly, in the latter study, while the authors reported a strong inhibitory effect of the CD137 agonist antibody on a T-cell dependent antigen (4-hydroxy-3-nitrophenylacetyl hapten conjugated with KLH), no effect on antibodies was observed against a T-cell independent antigen (4-hydroxy-3-nitrophenylacetyl hapten-ficoll complex) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), further supporting the involvement of T-cells in mCD137L-mediated inhibition of B-cell activation. In our study however both M2e- and NP-specific antibody responses are expected to be T-cell dependent (31, 32), and the exact mechanism underlying the mCD137L-mediated suppression of M2e- but not NP-specific antibody responses remains unclear. The intracellular localization of NP implies that NP-specific B-cells interact with NP after NP producing cells died and released the antigen, perhaps minimizing interference with surface-expressed CD137L.\u003c/p\u003e \u003cp\u003eIn conclusion, our work supports that incorporating CD137L in \u003cem\u003ecis\u003c/em\u003e within an mRNA\u0026ndash;LNP platform may offer a useful way to tune vaccine immunity toward strong, sustained T-cell responses while limiting antibody production against a defined T-cell\u0026ndash;dependent antigen. This vaccine strategy could be particularly advantageous against pathogens for which T-cells have been established as immune correlates of protection, and/or when antibody responses are undesirable, including infectious diseases for which antibody-dependent enhancement is an issue, as exemplified during Dengue vaccination or the early anti-RSV vaccines designs (reviewed in (33)).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant proteins and peptides\u003c/h2\u003e \u003cp\u003ePurified NP protein was purchased from Sino Biological (40776-V08B-100). M2e sequence used was MSLLTEVETPIRNEWGCRCNDSSD and was custom made by Genscript. NP\u003csub\u003e366\u0026ndash;374\u003c/sub\u003e peptide (TYQRTRALV) was purchased from Genscript. Alpha pool of 53 overlapping peptides from SARS-CoV-2 RBD B.1.1.7 was purchased from Genscript (RP30026). Peptide pool was reconstituted in DMSO (D2650, Sigma-Aldrich).\u003c/p\u003e \u003cp\u003ePurified SARS CoV 2 MBP-RBD and MBP-mCD137L recombinant proteins were produced by the Antibody Core Facility, LSI, NUS. Briefly, genes encoding SARS-CoV-2-RBD (aa 319\u0026ndash;591 of SARS-CoV-2 spike QVX50359.1) or murine CD137L (aa 104\u0026ndash;309 of NP_033430.1) were cloned into expression vector pHLmMBP-10 (Addgene) allowing expression of fusion proteins with N-terminal MBP and hexahistidine tags. MBP-RBD and MBP-mCD137L proteins were expressed in Expi293F cells (ThermoFisher Scientific, RRID: CVCL_D615) using Expi293 System. Culture supernatants were harvested on day 5 post transfection, and supernatants were incubated with nickel beads (Roche) at 4\u0026deg;C for 1 h. Nickel beads were equilibrated with binding buffer (25 mM Tris, 300 mM NaCl, pH 8.0) prior to incubation with culture supernatants. Supernatants containing beads were passed through cOmplete His-Tag Purification column (Roche) twice, and column was washed with binding buffer containing increasing concentrations of Imidazole. Proteins were eluted with elution buffer (25 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0), concentrated down and buffer-exchanged into 25 mM Tris, 300 mM NaCl, pH 8.0, and quantified by NanoDrop A280. Purity was assessed by standard denaturing polyacrylamide gel electrophoresis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMouse immunisation\u003c/h3\u003e\n\u003cp\u003eFor Flu vaccination model, 5\u0026ndash;6 week old female Balb/c mice (InVivos) were injected intramuscularly (i.m.) in the hind leg with 5 \u0026micro;g of M2-NP mRNA-LNP or 7.5 \u0026micro;g of CD137L-M2-NP mRNA-LNP in a total volume of 50 \u0026micro;l (in PBS) in a prime-boost regimen with 3-week interval. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of NUS under protocol number R23-0588. For SARS-CoV-2 immunization model, 6\u0026ndash;8 week old C57BL/6 mice male or female (single sex per experimental replicate) were injected twice at 3-week interval i.m. in the hind leg with 20 \u0026micro;g SARS CoV 2 MBP-RBD and 25 \u0026micro;L of AddaVax\u0026trade; adjuvant (vac-adx-10, InvivoGen) with or without 20 \u0026micro;g of mCD137L (in a final volume of 50 \u0026micro;L in PBS). All mice experiments were performed under IACUC protocol 231806 and in accordance to the National Advisory NACLAR guidelines.\u003c/p\u003e\n\u003ch3\u003eSerum collection\u003c/h3\u003e\n\u003cp\u003eBlood was collected under light anaesthesia (isoflurane 4% for induction, 1\u0026ndash;2% for maintenance. Blood was left to clot for 1 h at room temperature, centrifuged at 2,500 g for 10 min at 10\u0026deg;C to acquire serum. Serum was then heat inactivated at 56\u0026deg;C for 30 min and stored at -80\u0026deg;C before downstream ELISA.\u003c/p\u003e\n\u003ch3\u003ePreparation of splenocyte suspensions\u003c/h3\u003e\n\u003cp\u003eSpleens were harvested from euthanized mice and single cell suspensions were prepared by mechanical disruption and filtration through 40mm cell strainer in complete RPMI (cRPMI) (RPMI 1640 medium\u0026thinsp;+\u0026thinsp;10% FBS\u0026thinsp;+\u0026thinsp;1% Pen/Strep). Cell suspensions were centrifuged at 400 xg for 5 min at 10\u0026deg;C, red blood cells were lysed with Flow Cytometry Mouse Lyse Buffer (FC003, R\u0026amp;D Systems) at room temperature for 5 min. Cell suspensions were then quenched with cRPMI, centrifuged again at 400 xg for 5 min at 10\u0026deg;C and resuspended with cRPMI\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eFor M2e-and NP specific T-cell responses, freshly prepared splenocyte suspensions (2x10\u003csup\u003e6\u003c/sup\u003e) were seeded into U-bottom 96-well plates. A final concentration of 20 \u0026micro;g/mL of NP\u003csub\u003e366\u0026ndash;374\u003c/sub\u003e peptide or M2e peptide was added into each relevant well and the plate was incubated together with anti-CD107a antibody (BioLegend) (1:100 dilution) for 16 h at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Next, brefeldin A (1:100 dilution) (ThermoFisher Scientific) was added and incubated further for 3 h. For the positive control, a final concentration of 50 ng/mL Phorbol 12-myristate 13-acetate (PMA) (Invivogen,) and 1\u0026micro;g/mL of ionomycin (Invivogen) were added. For the negative control, 0.25% DMSO in cRPMI was added. The plate was incubated for 4 h at 37\u0026deg;C, 5% CO2 before the cells were washed with 1xPBS. The cells were stained with Near IR live-dead dye (1000x dilution) (ThermoFisher Scientific) for 15 min in the dark at 4\u0026deg;C. Next, the cells were incubated with FcR blocker (25x dilution) (Miltenyi Biotec) for 30 min at 4\u0026deg;C. For both panels, the cells were washed with FACs buffer before staining of cell surface markers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The cells were resuspended in fix/perm buffer (ThermoFisher Scientific) and incubated for 30 min in the dark at 4\u0026deg;C. The cells were then washed with permeabilization buffer (ThermoFisher Scientific) and stained for intracellular cytokines (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) for 30 min in the dark at 4\u0026deg;C. Final washes with permeabilization and then FACs buffer were done before flow cytometry analysis.\u003c/p\u003e \u003cp\u003eFor RBD specific T-cells, freshly prepared splenocyte suspensions (2x10\u003csup\u003e6\u003c/sup\u003e) were stimulated overnight at 37\u0026deg;C with SARS-CoV-2 RBD peptide pool at 2.5 \u0026micro;g/mL with mouse IL-2 at 30U/mL (212-12-20UG, PeproTech) in complete IMDM (cIMDM) (IMDM\u0026thinsp;+\u0026thinsp;10% FBS\u0026thinsp;+\u0026thinsp;1% Pen/Strep). Brefeldin A (420601, Biolegend) and Monensin (420701, Biolegend) diluted in cIMDM were next added and incubated at 37\u0026deg;C for 3 h. Splenocytes were then stained with Live-Dead Fixable Blue (L23105, Invitrogen) for 5 min at 4\u0026deg;C in the dark, and blocked with TruStain FcX\u0026trade; PLUS (anti-mouse CD16/32) (156604, Biolegend) in MACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) for 15 min. Splenocytes were then stained for extracellular T-cell markers (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) for 30 min at 4\u0026deg;C. Streptavidin-StarBright Violet (STAR210SBV515, Biorad) was added for B cell, Macrophage and NK cell exclusion gate. Cells were fixed with BD FACS\u0026trade; Lysing Solution (349202, BD) and then permeabilised with BD FACS\u0026trade; Permeabilizing Solution (340973, BD). After permeabilisation, splenocytes were blocked again before staining for intracellular T-cell markers (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) at 4\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003eFor M2e specific GC B cells, M2e-specific GC B cells were detected using biotinylated M2e in combination with fluorophore-conjugated SAv. Biotinylated M2e was multimerized at a 4:1 molar ratio with SAv-BV421 and 24 SAv-PE (BD Biosciences) respectively. SAv-FITC was used as decoy probe without biotinylated M2e to exclude cells with non-specific binding to SAv. After multimerization, M2e probe master mix was made by mixing the fluorophore-conjugated M2e probes with 5 \u0026micro;M free D-biotin (Thermo Fisher Scientific, B20656) to minimize cross-reactivity between probes, diluted in a 1:3 mixture of Brilliant Stain buffer (BD Biosciences) and FACS buffer (2% FBS\u0026thinsp;+\u0026thinsp;1 mM EDTA in PBS). Cells were then incubated with anti-CD16/CD32 Fc block (BD Biosciences) and eFluor 780 Fixable Viability Dye (Thermo Fisher Scientific) diluted in FACS buffer (1:200 and 1:1,000 respectively) for 20 min at 4\u0026deg;C. Cells were washed with FACS buffer and stained with respective decoy probe (Table S3) diluted in FACS buffer with 5 \u0026micro;M free D-biotin for 30 min at 4\u0026deg;C in the dark. Subsequently, cells were washed and stained with M2e probe master mix for 1h at 4\u0026deg;C in the dark. Thereafter, cells were washed and stained with GC B cell surface marker antibodies (Table S3) diluted in Brilliant Stain and FACS buffer for 30 min at 4\u0026deg;C in the dark.\u003c/p\u003e \u003cp\u003eFor RBD specific B cells, freshly prepared splenocytes (5x10\u003csup\u003e6\u003c/sup\u003e) were stained with Live-Dead Fixable Blue (L23105, Invitrogen) for 10 min at 4\u0026deg;C in the dark, then blocked with TruStain FcX\u0026trade; PLUS (anti-mouse CD16/32) (156604, Biolegend) in MACS buffer (1X PBS, 0.5% BSA, 2mM EDTA) for 15 min. RBD specific B cell subsets were detected by using biotinylated SARS-CoV-2 RBD protein (793906, Biolegend) multimerised with Streptavidin-PE-Fire 700 (405174, Biolegend) at molar ratio of 4:1 in a 50/50 mixture of PBS with 2% FBS and MACS buffer as previously described (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). After multimerization Streptavidin-StarBright Violet (STAR210SBV515, Biorad) was added as a decoy to gate ouT-cells that bind non-specifically to RBD. Then 5 \u0026micro;M free D-biotin (B20656; Thermo Fisher Scientific) was added to minimise probe cross reactivity. Cells were then stained with RBD probe mastermix for 1h at 4\u0026deg;C in the dark. After incubation, cells were then stained with the B cell markers listed in (Table S4) for 30 min at 4\u0026deg;C. Cells were then fixed with BD FACS\u0026trade; Lysing Solution (349202, BD).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eFor anti-M2e-specific antibody titres, 96-well plates were coated with M2e (150ng per well) and incubated overnight at 4\u0026deg;C. The plate was then washed three times with wash buffer (1x PBS, 0.05% Tween-20), then blocked with blocking buffer (1x PBS, 1% Bovine Serum Albumin for 1 h at 37\u0026deg;C. The plate was then washed once with wash buffer before adding serially diluted (two-fold) mice sera and incubated at 37\u0026deg;C for 1 h. The plate was washed thrice before the addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Bio-Rad, 170\u0026ndash;6516) at 1:3,000 (in 1% BSA). After incubation at 37\u0026deg;C for 1 h, the plates were washed three times. The o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich, P9187-50SET) was added to each well and incubated at room temperature for 10 min in the dark. H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added to each well to stop the reaction, and absorbance was read at 490 nm. Antibody titres were determined by non-linear regression as the reciprocal of the highest serum dilution with absorbance corresponding to three times the absorbance of blank wells.\u003c/p\u003e \u003cp\u003eFor anti-NP IgG, 96-well plates were coated with NP (150ng per well) and incubated overnight at 4\u0026deg;C. The plate was then washed three times with wash buffer (1x PBS, 0.05% Tween-20), then blocked with blocking buffer (1x PBS, 1% Bovine Serum Albumin for 1 h at 37\u0026deg;C. The plate was then washed once with wash buffer before adding serially diluted (two-fold) mice sera and incubated at 37\u0026deg;C for 1 h. The plate was then washed thrice before the addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Bio-Rad, 170\u0026ndash;6516) at 1:3,000 (in 1% BSA). After incubation at 37\u0026deg;C for 1 h, the plates were then washed three times. The o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich, P9187-50SET) was added to each well and incubated at room temperature for 10 min in the dark. H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003ewas added to each well to stop the reaction, and absorbance was read at 490 nm. Antibody titres were determined by non-linear regression as the reciprocal of the highest serum dilution with absorbance corresponding to three times the absorbance of blank wells.\u003c/p\u003e \u003cp\u003eFor anti-RBD IgG, MaxiSorp Immuno plates (442404, Thermo Scientific) were coated with purified SARS CoV 2 MBP-RBD at 10 \u0026micro;g/mL and incubated overnight at 4\u0026deg;C. Mouse sera were diluted 1:500 in antibody dilution buffer [1X PBS, 0.5% Bovine Serum Albumin (BSA) (SLCQ7756, Sigma-Aldrich), 0.05% Tween20 (P1379, Sigma-Aldrich)]. Plates were washed twice with washing buffer (1X PBS, 0.05% Tween20) before addition of diluted mouse sera (1:500 dilution) and incubated at 37\u0026deg;C for 1 h. Plates were then washed twice with washing buffer before addition of Goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Secondary antibody HRP (1:1000) (31430, Invitrogen) diluted in antibody dilution buffer and incubated at 37\u0026deg;C for 30 min. After incubation, the plates were washed twice with washing buffer and once with distilled water. TMB substrate (T8665, Sigma-Aldrich) was added, and plates were incubated in the dark for 6 min, reaction was stopped with 0.16 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (S5814, Sigma-Aldrich). Absorbance was read at 450 nm (reference 690nm) on a Tecan plate reader.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurrogate Virus Neutralization Test\u003c/h3\u003e\n\u003cp\u003ecPass\u0026trade; SARS-CoV-2 Neutralization Antibody Detection Kit (L00847-A, Genscript) was used to detect SARS-CoV-2 neutralizing antibodies in mouse sera (1:10 dilution) as per the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eData acquisition and analysis\u003c/h3\u003e\n\u003cp\u003eStained splenocytes were acquired on Novocyte Penteon (Agilent Technologies) and data analysis was performed on FlowJo (v.10.9.0). Gating strategies for each antibody panel are shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Figures and statistics were plotted using GraphPad Prism (v10.5.0).\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKS, MXL, DZWL, JLN, WL and EG performed the experiments.J.L.L. and E.N. produced the recombinant proteins.KS, HS, SA and GC designed the experiments, analysed the data and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe study was funded by the Programme for Research in Epidemic Preparedness And REsponse (PREPARE) awarded to HS (PREPARE-OC-VT-2022-008). Part of this work was also supported by A*STAR core funding and NMRC OF-IRG (MOH-001618-00) awarded to GC. We thank A*STAR IDL Pathogen Flow platform team, as well as A/P Paul Hutchinson and his team from the flow cytometry core facility at Life Sciences Institute, NUS for their invaluable help in this study. We also thank the Antibody Engineering Core Facility at LSI for their help in producing purified RBD and CD137L proteins.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll figure data is available as supplemental file 1. Other data are available upon request from lead authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoss P. 2022. The T cell immune response against SARS-CoV-2. Nat Immunol 23:186\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEggenhuizen PJ, Ooi JD. 2024. The Influence of Cross-Reactive T Cells in COVID-19. 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Mol Ther 32:2299\u0026ndash;2315.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9365361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9365361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCD137 engagement through CD137 ligand (CD137L) or agonistic antibodies has shown promising results in cancer immunotherapy. Here, we explored CD137L as an adjuvant to improve vaccine-induced T-cell mediated immunity. Here we show that including CD137L in \u003cem\u003ecis\u003c/em\u003e in mRNA vaccine significantly boosted T-cell responses, but not in protein vaccine. Furthermore, CD137L-containing vaccines induced lower antibody responses. 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