BCG enables protection against malaria through adjuvanting an Adenovirus-vectored vaccine

BCG enables protection against malaria through adjuvanting an Adenovirus-vectored vaccine | 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 BCG enables protection against malaria through adjuvanting an Adenovirus-vectored vaccine Sören Reinke, Marta Ulaszewska, Qi Su, Lee Sims, Romain Guyon, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7858925/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The BCG vaccine against TB offers broader non-specific health benefits, including reduced malaria infection. This may stem from its ability to epigenetically imprint innate immune cells, enhancing subsequent response to pro-inflammatory stimuli, coupled with intrinsic adjuvanticity through direct activation of cell surface PRRs. Here we investigate whether vaccinating with BCG in parallel with an adenoviral-vectored vaccine could be beneficial for protection against malaria in a mouse model. We show that combining the two vaccines broadens and strengthens the early innate cytokine repertoire, enabling strong CD8 + T cell-dependent protection. Our findings reveal co-immunisation with BCG and a viral vector as a promising strategy to combat both TB and malaria in double-endemic regions. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Vaccine Viral Vector AdHu5 BCG Malaria Figures Figure 1 Figure 2 Main text The tuberculosis (TB) vaccine Bacillus Calmette-Guérin (BCG) has well-documented off-target benefits against a broad range of infections, including malaria, sepsis, leprosy, SARS-CoV-2, and even as therapy in bladder cancer 1 . These non-specific effects are believed to be underpinned by epigenetic imprinting of myeloid cells 2 , resulting in enhanced secretion of pro-inflammatory cytokines such as TNF and IL-1β for up to a few months post BCG vaccination 3 . BCG-induced non-specific immunity against malaria 4-8 is of particular significance as the endemic geographic regions of TB and malaria largely overlap 9 . Field studies in sub-Saharan Africa have shown association between BCG vaccination and reduced malaria prevalence in children under 5, and seasonal reductions in all-cause neonatal mortality 9,10 . To date, the protective effect of BCG has not been investigated in the context of malaria vaccination. In view of its specific adjuvant-like properties through pathogen recognition receptor (PRR) activation, along with the off-target effect on malaria, it is relevant to consider vaccination strategies that administer malaria and BCG vaccines at the same time or in temporal proximity, to exploit the full potential of BCG in reducing both malaria and TB burden in double-endemic regions. This study explores the effect of BCG on the immune response elicited by an adenoviral-vectored malaria vaccine (human Adenovirus serotype 5, hereafter referred to as Ad), in a mouse model of malaria. The Ad construct encodes an immunodominant CD8 + T cell epitope Pb9 from Plasmodium berghei ( P. berghei ) circumsporozoite protein, which mediates protection against liver-stage malaria in this mouse model 11-13 . Mice were immunised with a low dose of Ad (offering no protection), BCG alone, or with Ad+BCG (injected at different sites), and challenged with P. berghei malaria parasites 2-4 weeks later (Fig. 1a). Mice vaccinated with Ad or BCG alone had no protection against malaria (Fig. 1b), whereas 80% of the mice that received both vaccines developed strong sterile immunity (Fig. 1b). This suggests that BCG is adjuvanting the adaptive, Pb9 CD8 + T cell driven, response against malaria either through innate immune imprinting or by directly adjuvanting the innate response to the Ad-vectored vaccine. To assess the role of myeloid cell imprinting 14 in protection, mice were vaccinated with BCG two or four weeks prior to Ad vaccination. These regimens resulted in a lower survival rate compared to Ad-BCG co-administration (Fig S1a), suggesting that the key element in protection against malaria in this model is the direct BCG-adjuvanting effect of the Ad vaccine. To mechanistically dissect the protective immune response and assess potential synergy between BCG and Ad in the context of liver-stage malaria, we phenotyped splenic and hepatic CD8 + T cells 15 and analysed antibody (Ab) responses after vaccination with Ad, BCG and Ad+BCG (Fig.1a). Of note, a reduced percentage of Pb9 + specific CD8 + T cells was observed in the spleens and livers of mice receiving Ad+BCG compared to Ad alone (Fig 1c-e, S1b, c), replicating our previous findings with adjuvanted Ad vaccine in malaria protection 12 . Vaccination with BCG also affected the distribution of Pb9 + CD8 + T cell memory sub-populations, resulting in a higher proportion of central memory T cells (Tcm) and reduction in effector memory T cells (Tem) that with Ad vaccine alone (Fig. 1d). Importantly, immunisation with BCG did not affect the CD8 + T cell surface density of the liver-homing cell marker CXCR6 16 (Fig S1b, c), which has previously been associated with protection against liver stage malaria in mice, and which we found to be more abundant on Pb9-specific T cells compared to the Pb9 - CD8 + T cell subset (Fig S1b, c). Given that the adaptive immunity against malaria generated by this vaccine is reliant solely on the Pb9 T cell epitope, the observed reduction in total antigen (Ag)-specific CD8 + T cells suggests that BCG-induced perturbation of T cell memory subsets may be of significance, with the protection driven by the type (rather than the total number) of Ag-specific CD8 + T cells. Antigen-specific Tcm, which were enriched after Ad+BCG vaccination, have also previously been demonstrated to enhance sterile protection in this model 12 . Indeed, the efficiency of the CD8 + T cell response relies on the quantity/density of the presented Ag, as well as the quality of co-stimulation by the myeloid antigen presenting cells which are targets of BCG activation 17 . Investigating the effect of Ad and BCG co-administration on generating B cells and Ab responses, we found a reduction in the total number of germinal centre B cells and plasma cells in mice that received Ad+BCG, compared to Ad alone (Fig. 1f-h). Likewise, immunisation with Ad+BCG led to a slight reduction in the overall numbers of plasmablasts and memory B cells, compared to Ad alone (Fig. S2a-c). To investigate if addition of BCG might have resulted in a weakened Ab response against the Ad vaccine, which encodes GFP as a surrogate Ag, we measured anti-GFP IgG titres post vaccination. Akin to our T cell data, additional immunisation with BCG did not affect Ag-specific IgG titres, indicating that despite a reduction in the number of circulating lymphocytes, BCG has the capacity to generate more effective B cells (Fig. 1i). In parallel, the addition of the Ad vaccine enhanced the anti-BCG humoral response, and increased seroconversion against BCG in the Ad+BCG group compared to BCG alone. (Fig. 1g, 1j). These findings suggest a reciprocal adjuvanting effect between Ad and BCG, indicating that simultaneous vaccination with BCG and viral-vectored vaccines could enhance protection against both TB and the disease targeted by the vectored vaccine. Activation of innate immunity by BCG is well documented 17 , and could play a key mechanistic role in the observed effect of BCG on the adaptive response to the Ad-vectored vaccine. We profiled the early innate responses to the three vaccination regimens (Ad, BGC, Ad+BCG), by analysing the cytokine secretion patterns at the site of injection (SOI), and systemically, in the serum and the spleen (Fig. 2a). There was a clear difference in systemically induced cytokines between Ad and BCG, with Ad inducing IFN-γ, IFN-α and CXCL-10 secretion, and BCG promoting production of CXCL1 and IL-6 (Fig. 2b, c, S3). This effect was particularly pronounced in the serum. Simultaneous vaccination with Ad and BCG resulted in a broader, merged, pattern of secreted cytokines (Fig 2b, S3), supporting the premise of BCG-promoted adjuvantation through expanding the pro-inflammatory cytokine spectrum. Locally, in the muscle (SOI), additional cytokines were induced by Ad (CCL5) or BCG (CCL2, IL-1β, GM-CSF, TNF, Fig. 2d, S3) which were not detected systemically. Interestingly, in Ad+BCG vaccinated mice, with Ad and BCG given into the opposite limbs, the vaccinated sites showed lower cytokine secretion compared to the respective single-vaccinated mice (Fig. 2d). However, this did not affect the cytokines on the systemic level, either qualitatively or quantitatively, as seen in the serum and the spleen (Fig. 2b, c, S3). Overall, our innate cytokine data are in line with previous findings from immunising with Ad 18 or BCG 19 alone, and reveal the induction of a pool of pro-inflammatory cytokines (IFN-γ, IFN-α, IL-6, IL-1β, TNF), chemo-attractants (CXCL1, CCL2, CCL5, CXCL10), and the colony-stimulating factor GM-CSF, when both vaccines are given at the same time. To corroborate these findings in a human system, we utilised human macrophages derived from peripheral-blood CD14 + monocytes by M-CSF stimulation. Activation of the NLRP3 inflammasome after in vitro stimulation with Ad, BCG or Ad+BCG (Fig 2e) was measured as a key indicator of adjuvanticity 20 and BCG-mediated imprinting 21 . Direct TLR stimulation of myeloid cells induces NFkB signalling, promoting TNF secretion and the expression and post-translational modifications of the NLRP3 inflammasome 20 . TLR priming also strongly induces the expression of pro-IL-1β, ready for inflammasome-mediated processing into the active form. Full inflammasome activation requires a second signal, typically delivered from pathogen or vaccine uptake, leading to the secretion of IL-1β and IL-18, and pro-inflammatory cell death 20 . Interestingly, in vitro, Ad and BCG individually induced low amounts of secreted IL-1β, and when combined led to a strong IL-1β secretion across a range of doses (Fig 2f). AdHu5 vector is known as a poor inducer of secreted IL-1β 22 , indicating either lack of induction of pro-IL-1β, or absence of inflammasome activation. BCG can induce transcriptional upregulation of pro-IL-1β but only limited secretion of the active cytokine, in line with its recognised role as a modest activator of the inflammasome pathway 23 . Our data support this, demonstrating secreted IL-1β in the BCG- but not the Ad-vaccinated muscle (Fig 2c, S3). Similarly, combined Ad+BCG stimulation strongly activated TNF secretion, which is induced by TLR signalling and independent of the inflammasome activation (Fig 2f). Inflammasome-dependent cell death, measured by the release of cytosolic enzyme lactate dehydrogenase (LDH), was found to be vaccine dose-dependent and only elevated at the highest concentrations of Ad+BCG (Fig 2f). These findings demonstrate that, in human macrophages, Ad and BCG strongly synergise in TLR and inflammasome pathway activation which neither of the vaccines can achieve individually. As TLR and inflammasome activation are hallmarks of potent adjuvanticity 20 - a key contributor to effective vaccination - engagement of these pathways is likely to be beneficial for both vaccines. In summary, we demonstrate that combining BCG with a viral-vectored malaria vaccine generates a broader innate immune response and enables protection against malaria challenge. Simultaneous administration of both vaccines induces a potent inflammatory cytokine profile, capable of supporting protective CD8 + T cells in this challenge model. Recent studies in mice have shown that intravenously administered BCG can protect against SARS‑CoV‑2 via CD4 + T cell mediated IFN-γ secretion, through imprinting of myeloid and epithelial cells for enhanced antiviral immunity 24,25 . In clinical trials, previous administration of BCG has been found to enhance the immune response to RNA- or viral vector-based SARS-CoV-2 vaccines 26,27 . Consequently, our findings broaden the portfolio of advantageous applications of the BCG vaccine, identifying co-administration of BCG and malaria vaccines as a potentially promising strategy to combat both TB and malaria in double-endemic regions. Methods Mice and Immunisations For all experiments, mice were purchased from Envigo, UK. Animals were maintained at the Centre for Human Genetics, University of Oxford, housed under Specific Pathogen Free (SPF) conditions in individually ventilated cages, with constant temperature and humidity and a 12 hour light/dark cycle, and in accordance with the recommendations of the UK Animals (Scientific Procedures) Act 1986 and ARRIVE guidelines. Protocols were approved by the University of Oxford Animal Care and Ethical Review Committee for use under Project Licenses P9804B4F1 and PP0984913, granted by the UK Home Office. Anaesthetised (using vaporized IsoFlo ® ) female BALB/c mice (BALB/cOlaHsd) mice, aged 7–10-weeks, were immunised intramuscularly (i.m.) with a total volume of 50 μL in the tibialis muscle. In the initial experiments BCG was administered intradermally (i.d.) into the ear (2 x 25 μL each site). Comparison with i.m. administration showed no difference in protection against malaria and BCG was given i.m. in subsequent experiments. Each vaccine dose contained 0.5 x 10 5 CFU BCG, or 5 x 10 7 i.u. AdHu5-TIPeGFP. Sporozoite production Plasmodium berghei (ANKA strain clone 234) sporozoites were produced as previously described 28 . Starved female Anopheles stephensi mosquitoes were fed for approximately 10 min on BALB/c mice infected with P. berghei parasites. The mosquitoes were then maintained on Fructose/PABA solution at 19–21°C in a humidified incubator on 12 h light/dark cycle. Approximately 21 days after feeding, the mosquitoes were dissected and the salivary glands removed. While keeping on ice, the glands were placed in RPMI-1640 and disrupted with a tissue homogeniser to release the sporozoites. The sporozoites were counted using a haemocytometer, while keeping on ice to preserve viability, and immediately proceeded to malaria challenge. Malaria challenge Malaria challenge was performed as described previously 28 . For all experiments 1,000 P. berghei sporozoites (described above) were injected intravenously in a total volume of 100 μL into the lateral tail vein of each mouse. From day 5 post challenge the mice were monitored for infection by thin-film blood smear (fixed in methanol and stained in 10% Giemsa for 30 min). Mice were sacrificed when >1% parasitaemia was observed. If no parasites were detected on day 12 after challenge, mice were considered sterilely protected. Ex-vivo IFN-γ splenocyte ELISpot ELISpot assays were performed using the IFN-γ ELISpot (kit 3321-2A, Mabtech, UK) as described previously 28 . In brief, two weeks after immunisation mice were culled and spleens removed. Single cell suspensions were prepared by homogenising and straining the spleens followed by brief incubation in ACK lysis buffer to remove erythrocytes. Cells were washed with PBS, pelleted, resuspended in complete α-MEM and counted using the CASY cell counter. MultiScreen-IP 0.45 μm sterile plates were coated with 5 μg/ml rat anti-mouse interferon gamma mAb AN18 diluted in carbonate-bicarbonate buffer overnight at 4 °C. Plates were then blocked for at least one hour at 37 °C with complete α-MEM. Cells were plated and stimulated for 18–20 h with 1 μg/mL of Pb9 peptide. Plates were washed and incubated with 1 μg/mL biotinylated anti-mouse-IFNγ mAb R46A2, followed by incubation with 1 μg/mL streptavidin alkaline phosphatase. Spots were developed by addition of 50 μL per well of colour development buffer and counted using ELISpot software (AID). Results are expressed as spot forming cells (SFC) per million splenocytes after subtracting background responses in unstimulated wells. Flow Cytometry Phenotypic analysis of CD8 + T cells was performed by surface marker staining using the following fluorescently labelled antibodies: PE-Cy7-CD11a (BD Bioscience cat #558191), PE-CD186 (Biolegend cat #151104), BV711-CD69 (Biolegend cat #104537), BV650-CD44 (Biolegend cat #103049), BV605-CD62L (Biolegend cat #104437), BV421-KLRG1 (Biolegend cat #138413), AF700-CD8 (Biolegend cat #100729). Non-specific Ab binding was prevented by incubation with anti-CD16/CD32 Fcγ III/II Receptor (BD Pharmingen, UK) prior to the surface staining. The Pb9 tetramer was provided by the NIH tetramer facility (MHC tetramer core facility, Emory University Vaccine Center, Atlanta, USA) using SYIPSAEKI peptide. Live and dead cells were distinguished using LIVE/DEAD TM Fixable Aqua Dead Stain Kit (ThermoFisher Scientific, UK). To define subpopulation distribution within the Pb9 + compartment (Figure 1d, e) cells were gated on singlets, size, CD8 + , Pb9 + , and subsequent definition of subpopulations as depicted in Figure S1d without gating on CD44 + /CD11a + as Pb9 + cells were generally positive for these markers. Flow cytometric analyses were performed on LSRII (BD Biosciences, UK) and data analysed with FlowJo software. For B cell profiling, freshly harvested splenocytes were seeded in 96-well plates at a density of ~1.5x10 6 cells per well in chilled FACS buffer. Cells were washed and stained with diluted nIR Zombie L/D stain (Biolegend cat #423105) and incubated for 15 minutes at room temperature. After incubation, cells were washed and stained with a cocktail of the following antibodies diluted in FACS buffer: BV421-CD19 (Biolegend cat #115549), BV605-B220 (Biolegend cat #130244), BV786-CD95 (BD Bioscience cat #740716), PerCPCy5.5-GL7 (Biolegend cat #144609), PECy5-CD38 (Invitrogen cat #15-0381-82), PECy7-CD138 (Biolegend cat #142513), AF700-IgD (Biolegend cat #405729), APCCy7-CD3 (Biolegend cat #100222), APCCy7-F4/80 (Biolegend cat #123117). Cells were subsequently washed and transferred to FACS tubes for flow cytometric analyses on a Fortessa (BD Biosciences, UK). Total IgG ELISA ELISAs to detect antibodies against eGFP (expressed by AdHu5-TIPeGFP) or total BCG were performed as previously described 28 . Serum was obtained by collecting blood from the lateral tail vein in a microcuvette tube. Blood was allowed to clot at 4 °C overnight before centrifugation at 13,000 rpm for 4 min and sera removed and stored at −20 °C until use. For total IgG ELISAs, Nunc-Immuno Maxisorp 96 well plates were coated with 1 μg/mL eGFP in carbonate-bicarbonate coating buffer or 10 5 CFU BCG per well overnight at 4 °C. Plates were washed with PBS-Tween (0.05% v/v) and blocked with 2% BSA in PBS-Tween for 1 h at RT. Sera were diluted appropriately between 1:200 and 1:3,200 (depending on Ag and time point) in 1% BSA in PBS-Tween and added to plate in duplicates. Plates were incubated for 2 h at room temperature and then washed as before. Fc-specific goat anti-mouse IgG conjugated to alkaline phosphatase (AP) (1 in 5,000, Sigma-Aldrich, A1418) was added for 1 h at room temperature (eGFP) or overnight at 4°C (BCG). Following a final wash, plates were developed by adding p -nitrophenylphosphate at 1 mg/mL in diethanolamine buffer and OD was read at 405 nm. Total IgG concentrations against eGFP or BCG in sera were calculated by interpolation against a standard curve generated from a serum pool of previously AdHu5-TIPeGFP or BCG immunised mice. Sera from naive mice were used as negative control. ELISA for detection of cytokines in serum, spleen, and muscle (SOI) Tissue cytokines were assessed as described previously 29 . To detect innate cytokines in serum, spleen and muscle (SOI) samples, mice were immunised with AdHu5-TIPeGFP and/or BCG as described above. At specific time points mice were sacrificed, and tissues harvested. Blood samples were allowed to clot for 2 h before centrifugation at 13,000 rpm for 4 min; sera were stored at −80 °C until use. Muscle and spleen samples were weighed, transferred into PBS supplemented with protease inhibitor (Roche, 11873580001), and dissociated in gentleMACS M Tubes using the gentleMACS Octo Dissociator. Cell suspensions were stored at −80°C. Before use, samples were thawed, clarified by centrifugation at 10,000 rpm for 5 min, and supernatants analysed for cytokines and chemokines using the mouse MU Anti-Virus Response Panel flow assay kit (LEGENDplex, Biolegend) according to the manufacturer’s instructions. LSR Fortessa (BD) flow cytometer was used to assess fluorescence intensity of beads and data analysed using LEGENDplex Data Analysis Software Suite. Human Monocyte Derived Macrophages (HMDM) Assays on Human Monocyte Derived Macrophages were performed as previously described 29 . In brief, peripheral blood mononuclear cells were isolated using Ficoll gradient from healthy donors from NHS Oxford blood bank (REC approval 11/H0711/7). Magnetic beads (eBioscience, 8802-6834-74) were used to positively select CD14 + monocytes which were differentiated into macrophages by culturing for 7 days with M-CSF (100 ng/mL, BioLegend, 574808). Cells were cultured in RPMI (Fisher, 21870076) with 10% FBS (Life Technologies, 16000044) and 1x Pen/Strep/Glutamine (Fisher, 10378016) at 37 °C with 5% CO 2 , and supplemented with fresh media containing 100 ng/mL M-CSF on day 2 and day 5. After day 7 of differentiation, cells were re-plated (70,000 cells per well in flat bottom 96-well plates in 100 μL complete RPMI) and on day 8 cells were stimulated for inflammasome-activation experiments. Differentiated HMDMs were plated at a density of 0.7x10 6 cells/mL complete RPMI and stimulated for 24 h with AdHu5-TIPeGFP and/or BCG at concentrations indicated in the Figures. Culture media was replaced with 100 µL OptiMEM (Fisher, 31985062) immediately before stimulation. After cell stimulation, cell supernatants were collected for analysis. Secretion of IL-1β and TNF was measured in cell-free supernatants using ELISA (Thermo Fisher, 88-7261-77 and 88-7346-88) according to manufacturer’s instructions. Cellular viability was assessed using cell culture supernatants and the Cytox96 nonradioactive cytotoxicity assay (Promega, G1780). Statistical analysis Statistical analysis was performed using Prism software (V10). Statistical tests applied and pairwise comparisons with correction for multiple testing are indicated in the Figure legends. In all experiments ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Non-significant differences (n.s.) are generally not indicated. Declarations Funding Declaration This work was funded in part by the MRC Confidence in Concept Award MC_PC_16056 (A.M.); VaxHub Global grant, Department of Health and Social Care using UK Aid funding, with support from EPSRC as part of the UK Vaccine Network (UKVN), a UK Aid programme to develop vaccines for diseases with epidemic potential in low and middle-income countries (LMICs) (A.M); Chan Zuckerberg Initiative grant 2020-217289 (A.M., J.F.), UK Medical Research Council MR/Y004450/1 (J.F.); NIHR Biomedical Research Centre, Inflammation Across Tissues Theme (J.F.); the Kennedy Trust for Rheumatology Research KENN212202 (J.S.B.), Medical Research Council MR/W001217/1 (J.S.B.); and Kennedy Trust for Rheumatology Research Studentship (S.Q.), The Clarendon Fund in partnership with an Oxford-St Cross E.P. Abraham Scholarship and Nuffield Department of Clinical Medicine Studentship (L.S.), Michelson Prize 2022 (R.G.). S.R. was funded in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 495054088). Acknowledgments We thank Elena Stylianou for sharing the expertise on growing BCG and intradermal injections, and Mariya Mykhaylyk for lab-keeping assistance. H2-Kd P. berghei CSP 245-253 SYIPSAEKI tetramer was kindly provided by the NIH Tetramer Core Facility (contract number 75N93020D00005). Illustrations were created with BioRender.com. Author Contribution (CRediT) Conceptualization: JRF, AM Methodology: SR, SQ, LS, JSB, AM Validation: SR, JRF, AM Formal analysis: SR, RG, JRF Investigation: SR, MU, SQ, LS, RG, AT, JRF, AM Resources: JSB, AM Data Curation: SR, JRF, AM Writing - Original Draft: SR Writing - Review & Editing: SR, MU, SQ, LS, RG, JSB, JRF, AM Visualization: SR Supervision: AM Project administration: JSB, JRF, AM Funding acquisition: SR, JSB, AM Declaration of Interest The authors declare no competing interests. References Jurczak, M. & Druszczynska, M. 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Supplementary Files MilicicBCGAdSupplementaryMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Dec, 2025 Reviews received at journal 07 Dec, 2025 Reviewers agreed at journal 22 Nov, 2025 Reviews received at journal 15 Nov, 2025 Reviewers agreed at journal 09 Nov, 2025 Reviewers invited by journal 07 Nov, 2025 Editor assigned by journal 06 Nov, 2025 Submission checks completed at journal 24 Oct, 2025 First submitted to journal 14 Oct, 2025 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. We do this by developing innovative software and high quality services for the global research community. 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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-7858925","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":532792568,"identity":"31dcc097-7b33-4bdc-9b9e-6bb7556bb741","order_by":0,"name":"Sören Reinke","email":"","orcid":"","institution":"The Jenner Institute, University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Sören","middleName":"","lastName":"Reinke","suffix":""},{"id":532792569,"identity":"36891d68-99bf-4458-8c54-737946e94c4a","order_by":1,"name":"Marta Ulaszewska","email":"","orcid":"","institution":"The Jenner Institute, University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Ulaszewska","suffix":""},{"id":532792570,"identity":"3ab117c1-c2fc-4cd5-9a7c-0c2a9960e7b6","order_by":2,"name":"Qi Su","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Su","suffix":""},{"id":532792571,"identity":"44e4ac18-f700-495c-a252-9cf10c2cb054","order_by":3,"name":"Lee Sims","email":"","orcid":"","institution":"The Jenner Institute, University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Lee","middleName":"","lastName":"Sims","suffix":""},{"id":532792573,"identity":"3efb5118-5f17-46be-81d1-15caa3d4009f","order_by":4,"name":"Romain Guyon","email":"","orcid":"","institution":"The Jenner Institute, University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Romain","middleName":"","lastName":"Guyon","suffix":""},{"id":532792574,"identity":"d3e2e783-3dc9-4911-a3e5-c99b6e60a07d","order_by":5,"name":"Adam Truby","email":"","orcid":"","institution":"The Jenner Institute, University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Truby","suffix":""},{"id":532792575,"identity":"d9cecd42-821a-467f-9d7b-6d0ffb863167","order_by":6,"name":"Jelena S. 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08:22:44","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99554,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7858925/v1/13124d6b4a32671d1f900ded.html"},{"id":94080665,"identity":"2aa2bb54-4977-4fe9-952e-d43ff6507309","added_by":"auto","created_at":"2025-10-22 08:30:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":914936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAd+BCG co-administration enables protection against malaria in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Summary of experimental design, indicating corresponding figure panels. Vaccine dose per mouse: 0.5x10\u003csup\u003e5\u003c/sup\u003e CFU for BCG (i.d. or i.m.) and 5x10\u003csup\u003e7\u003c/sup\u003e i.u. for AdHu5-TIPeGFP (i.m.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Survival post-malaria challenge: BALB/c mice were vaccinated and challenged as in (a). Parasitaemia was assessed by daily blood smears, with 1% parasitaemia taken as irreversible malaria infection. Ad+BCG vs. Ad or BCG alone: p \u0026lt; 0.01, pairwise fisher test with Bonferroni correction (n ≥ 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e IFN-γ producing CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleen two weeks post-vaccination, restimulated with the Pb9 peptide for 20 h ex vivo. Data show the mean + SEM (n = 8); one-way ANOVA with Bonferroni’s correction for multiple comparisons; **p\u0026nbsp;\u0026lt;\u0026nbsp;0.01; ****p\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d and e)\u003c/strong\u003e T cell phenotyping in spleen and liver after vaccination and harvest as in (a). Left-hand panels show percentage of antigen (Ag) specific CD8\u003csup\u003e+\u003c/sup\u003e T cells 3 or 4 weeks after vaccination. Right-hand panels show the distribution of T cell subpopulations within the Pb9\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e+\u003c/sup\u003e compartment. Gating strategy as in figure S1d. Mean + SEM (n = 6); 2-way ANOVA with Bonferroni’s correction for multiple comparisons; *p\u0026nbsp;\u0026lt;\u0026nbsp;0.05; **p\u0026nbsp;\u0026lt;\u0026nbsp;0.01; ***p\u0026nbsp;\u0026lt;\u0026nbsp;0.001; ****p\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f – h)\u003c/strong\u003e Total B cell phenotyping of splenocytes after vaccination and harvest as in (a). \u003cstrong\u003e(f)\u003c/strong\u003e Representative FACS plots of CD95\u003csup\u003e+\u003c/sup\u003e GL7\u003csup\u003e+\u003c/sup\u003e germinal centre B cell populations, showing percentages of live IgD\u003csup\u003e-\u003c/sup\u003e, lin\u003csup\u003e-\u003c/sup\u003e, CD138\u003csup\u003e-\u003c/sup\u003e, B220\u003csup\u003e+\u003c/sup\u003e lymphocytes. \u003cstrong\u003e(g)\u003c/strong\u003e Quantification of data presented in (f). Percentages of live IgD\u003csup\u003e-\u003c/sup\u003e, lin\u003csup\u003e-\u003c/sup\u003e lymphocytes. Each symbol represents an individual mouse (n = 8). \u003cstrong\u003e(h)\u003c/strong\u003e Quantification of plasma B cells after vaccination (n = 8). \u003cstrong\u003e(f-h)\u003c/strong\u003e Gating strategy as in figure S2d. Median + replicates; one-way ANOVA with Bonferroni’s correction for multiple comparisons; *p\u0026nbsp;\u0026lt;\u0026nbsp;0.05; **p\u0026nbsp;\u0026lt;\u0026nbsp;0.01; ***p\u0026nbsp;\u0026lt;\u0026nbsp;0.001; ****p\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i)\u003c/strong\u003e Serum anti-GFP IgG titres were measured by ELISA at the indicated time-points post immunisation; GFP is encoded by the Ad construct and used as a surrogate vaccine antigen. Each symbol represents an individual mouse (n = 8). Dashed line indicates the limit of detection. The panel on the right shows the anti-GFP antibody (Ab) titres over 4 weeks following vaccination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(j)\u003c/strong\u003e Serum anti-BCG IgG titres were measured by ELISA at the indicated time-points post immunisation. Each symbol represents an individual mouse (n = 8). Dashed line indicates the limit of detection. The panel on the right shows the anti-BCG Ab titres over 4 weeks post-vaccination. Testing for seroconversion at week 4 using Barnard's Unconditional Test;\u0026nbsp;*p\u0026nbsp;\u0026lt;\u0026nbsp;0.05.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7858925/v1/27be7c5a000fb5f0252ed28f.png"},{"id":94073854,"identity":"2790f6a9-96a3-4f8f-a357-435e232f688f","added_by":"auto","created_at":"2025-10-22 08:22:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":565417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCG broadens the systemic innate cytokine milieu\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Summary of the experimental design for data in (b-d). Vaccine dose per mouse: 0.5x10\u003csup\u003e5\u003c/sup\u003e CFU for BCG and 5x10\u003csup\u003e7\u003c/sup\u003e i.u. for AdHu5-TIPeGFP. In the Ad+BCG group, Ad was injected into the right leg and BCG into the left leg.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b - d)\u003c/strong\u003e Indicated cytokines were measured using LEGENDplex\u003csup\u003eTM\u003c/sup\u003e MU Anti-Virus Response Panel. Heatmaps show relative cytokine levels detected in b) the serum, c) the spleen, and d) the ipsi- and contralateral muscle as the site of immunisation (SOI) for each vaccine (n = 6). Ad alone and BCG alone: contralateral leg received no vaccine, Ad+BCG: each leg received one of the vaccines. Individual cytokine kinetics are shown in figure S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Summary of the experimental design for data in f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e HMDMs were stimulated with Ad and/or BCG for 24 h at the indicated MOI/CFU. IL-1β and TNF secretion in supernatants was measured by ELISA. LDH release as a measure of lytic cell death was quantified using a colorimetric assay and is depicted as a percentage of the lysed positive control. Representative data from two independent experiments, showing stimulation triplicates. One-way ANOVA with Bonferroni’s multiple comparison; *p\u0026nbsp;\u0026lt;\u0026nbsp;0.05; **p\u0026nbsp;\u0026lt;\u0026nbsp;0.01; ***p\u0026nbsp;\u0026lt;\u0026nbsp;0.001; ****p\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7858925/v1/9963dbe7ecd5e6a1addcc09d.png"},{"id":94128388,"identity":"91997236-1525-4bfb-9fd4-2e80d83bf83d","added_by":"auto","created_at":"2025-10-22 16:55:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1828644,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7858925/v1/db5bd989-5086-479c-9c31-a438091b69e8.pdf"},{"id":94073848,"identity":"e2fcfc31-429b-48a6-bc34-a9c305cc4f45","added_by":"auto","created_at":"2025-10-22 08:22:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2168958,"visible":true,"origin":"","legend":"","description":"","filename":"MilicicBCGAdSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7858925/v1/9245827ac794e32cb5c0c46b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"BCG enables protection against malaria through adjuvanting an Adenovirus-vectored vaccine","fulltext":[{"header":"Main text","content":"\u003cp\u003eThe tuberculosis (TB) vaccine Bacillus Calmette-Gu\u0026eacute;rin (BCG) has well-documented off-target benefits against a broad range of infections, including malaria, sepsis, leprosy, SARS-CoV-2, and even as therapy in bladder cancer\u003csup\u003e1\u003c/sup\u003e. These non-specific effects are believed to be underpinned by epigenetic imprinting of myeloid cells\u003csup\u003e2\u003c/sup\u003e, resulting in enhanced secretion of pro-inflammatory cytokines such as TNF and IL-1\u0026beta; for up to a few months post BCG vaccination\u003csup\u003e3\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBCG-induced non-specific immunity against malaria\u003csup\u003e4-8\u003c/sup\u003e is of particular significance as the endemic geographic regions of TB and malaria largely overlap\u003csup\u003e9\u003c/sup\u003e. Field studies in sub-Saharan Africa have shown association between BCG vaccination and reduced malaria prevalence in children under 5, and seasonal reductions in all-cause neonatal mortality\u003csup\u003e9,10\u003c/sup\u003e. To date, the protective effect of BCG has not been investigated in the context of malaria vaccination. In view of its specific adjuvant-like properties through pathogen recognition receptor (PRR) activation, along with the off-target effect on malaria, it is relevant to consider vaccination strategies that administer malaria and BCG vaccines at the same time or in temporal proximity, to exploit the full potential of BCG in reducing both malaria and TB burden in double-endemic regions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study explores the effect of BCG on the immune response elicited by an adenoviral-vectored malaria vaccine (human Adenovirus serotype 5, hereafter referred to as Ad), in a mouse model of malaria. The Ad construct encodes an immunodominant CD8\u003csup\u003e+\u003c/sup\u003e T cell epitope Pb9 from \u003cem\u003ePlasmodium berghei\u003c/em\u003e (\u003cem\u003eP. berghei\u003c/em\u003e) circumsporozoite protein, which mediates protection against liver-stage malaria in this mouse model\u003csup\u003e11-13\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMice were immunised with a low dose of Ad (offering no protection), BCG alone, or with Ad+BCG (injected at different sites), and challenged with \u003cem\u003eP. berghei\u003c/em\u003e malaria parasites 2-4 weeks later (Fig. 1a). Mice vaccinated with Ad or BCG alone had no protection against malaria (Fig. 1b), whereas 80% of the mice that received both vaccines developed strong sterile immunity (Fig. 1b). This suggests that BCG is adjuvanting the adaptive, Pb9 CD8\u003csup\u003e+\u003c/sup\u003e T cell driven, response against malaria either through innate immune imprinting or by directly adjuvanting the innate response to the Ad-vectored vaccine. To assess the role of myeloid cell imprinting\u003csup\u003e14\u003c/sup\u003e in protection, mice were vaccinated with BCG two or four weeks prior to Ad vaccination. These regimens resulted in a lower survival rate compared to Ad-BCG co-administration (Fig S1a), suggesting that the key element in protection against malaria in this model is the direct BCG-adjuvanting effect of the Ad vaccine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo mechanistically dissect the protective immune response and assess potential synergy between BCG and Ad in the context of liver-stage malaria, we phenotyped splenic and hepatic CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e15\u003c/sup\u003e and analysed antibody (Ab) responses after vaccination with Ad, BCG and Ad+BCG (Fig.1a). Of note, a reduced percentage of Pb9\u003csup\u003e+\u003c/sup\u003e specific CD8\u003csup\u003e+\u003c/sup\u003e T cells was observed in the spleens and livers of mice receiving Ad+BCG compared to Ad alone (Fig 1c-e, S1b, c), replicating our previous findings with adjuvanted Ad vaccine in malaria protection\u003csup\u003e12\u003c/sup\u003e. Vaccination with BCG also affected the distribution of Pb9\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cell memory sub-populations, resulting in a higher proportion of central memory T cells (Tcm) and reduction in effector memory T cells (Tem) that with Ad vaccine alone (Fig. 1d). Importantly, immunisation with BCG did not affect the CD8\u003csup\u003e+\u003c/sup\u003e T cell surface density of the liver-homing cell marker CXCR6\u003csup\u003e16\u003c/sup\u003e (Fig S1b, c), which has previously been associated with protection against liver stage malaria in mice, and which we found to be more abundant on Pb9-specific T cells compared to the Pb9\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cell subset (Fig S1b, c). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that the adaptive immunity against malaria generated by this vaccine is reliant solely on the Pb9 T cell epitope, the observed reduction in total antigen (Ag)-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells suggests that BCG-induced perturbation of T cell memory subsets may be of significance, with the protection driven by the type (rather than the total number) of Ag-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells. Antigen-specific Tcm, which were enriched after Ad+BCG vaccination, have also previously been demonstrated to enhance sterile protection in this model\u003csup\u003e12\u003c/sup\u003e. Indeed, the efficiency of the CD8\u003csup\u003e+\u003c/sup\u003e T cell response relies on the quantity/density of the presented Ag, as well as the quality of co-stimulation by the myeloid antigen presenting cells which are targets of BCG activation\u003csup\u003e17\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigating the effect of Ad and BCG co-administration on generating B cells and Ab responses, we found a reduction in the total number of germinal centre B cells and plasma cells in mice that received Ad+BCG, compared to Ad alone (Fig. 1f-h). Likewise, immunisation with Ad+BCG led to a slight reduction in the overall numbers of plasmablasts and memory B cells, compared to Ad alone (Fig. S2a-c). To investigate if addition of BCG might have resulted in a weakened Ab response against the Ad vaccine, which encodes GFP as a surrogate Ag, we measured anti-GFP IgG titres post vaccination. Akin to our T cell data, additional immunisation with BCG did not affect Ag-specific IgG titres, indicating that despite a reduction in the number of circulating lymphocytes, BCG has the capacity to generate more effective B cells (Fig. 1i). In parallel, the addition of the Ad vaccine enhanced the anti-BCG humoral response, and increased seroconversion against BCG in the Ad+BCG group compared to BCG alone. (Fig. 1g, 1j). These findings suggest a reciprocal adjuvanting effect between Ad and BCG, indicating that simultaneous vaccination with BCG and viral-vectored vaccines could enhance protection against both TB and the disease targeted by the vectored vaccine.\u003c/p\u003e\n\u003cp\u003eActivation of innate immunity by BCG is well documented\u003csup\u003e17\u003c/sup\u003e, and could play a key mechanistic role in the observed effect of BCG on the adaptive response to the Ad-vectored vaccine. We profiled the early innate responses to the three vaccination regimens (Ad, BGC, Ad+BCG), by analysing the cytokine secretion patterns at the site of injection (SOI), and systemically, in the serum and the spleen (Fig. 2a). There was a clear difference in systemically induced cytokines between Ad and BCG, with Ad inducing IFN-\u0026gamma;, IFN-\u0026alpha;\u0026nbsp;and CXCL-10 secretion, and BCG promoting production of CXCL1 and IL-6 (Fig. 2b, c, S3). This effect was particularly pronounced in the serum. Simultaneous vaccination with Ad and BCG resulted in a broader, merged, pattern of secreted cytokines (Fig 2b, S3), supporting the premise of BCG-promoted adjuvantation through expanding the pro-inflammatory cytokine spectrum. Locally, in the muscle (SOI), additional cytokines were induced by Ad (CCL5) or BCG (CCL2, IL-1\u0026beta;, GM-CSF, TNF, Fig. 2d, S3) which were not detected systemically. Interestingly, in Ad+BCG vaccinated mice, with Ad and BCG given into the opposite limbs, the vaccinated sites showed lower cytokine secretion compared to the respective single-vaccinated mice (Fig. 2d). However, this did not affect the cytokines on the systemic level, either qualitatively or quantitatively, as seen in the serum and the spleen (Fig. 2b, c, S3). Overall, our innate cytokine data are in line with previous findings from immunising with Ad\u003csup\u003e18\u003c/sup\u003e or BCG\u003csup\u003e19\u003c/sup\u003e alone, and reveal the induction of a pool of pro-inflammatory cytokines (IFN-\u0026gamma;, IFN-\u0026alpha;, IL-6, IL-1\u0026beta;, TNF), chemo-attractants (CXCL1, CCL2, CCL5, CXCL10), and the colony-stimulating factor GM-CSF, when both vaccines are given at the same time.\u003c/p\u003e\n\u003cp\u003eTo corroborate these findings in a human system, we utilised human macrophages derived from peripheral-blood CD14\u003csup\u003e+\u003c/sup\u003e monocytes by M-CSF stimulation. Activation of the NLRP3 inflammasome after in vitro stimulation with Ad, BCG or Ad+BCG (Fig 2e) was measured as a key indicator of adjuvanticity\u003csup\u003e20\u003c/sup\u003e and BCG-mediated imprinting\u003csup\u003e21\u003c/sup\u003e. Direct TLR stimulation of myeloid cells induces NFkB signalling, promoting TNF secretion and the expression and post-translational modifications of the NLRP3 inflammasome\u003csup\u003e20\u003c/sup\u003e. TLR priming also strongly induces the expression of pro-IL-1\u0026beta;, ready for inflammasome-mediated processing into the active form. Full inflammasome activation requires a second signal, typically delivered from pathogen or vaccine uptake, leading to the secretion of IL-1\u0026beta;\u0026nbsp;and IL-18, and pro-inflammatory cell death\u003csup\u003e20\u003c/sup\u003e. Interestingly, in vitro, Ad and BCG individually induced low amounts of secreted IL-1\u0026beta;, and when combined\u0026nbsp;led to a strong IL-1\u0026beta;\u0026nbsp;secretion across a range of doses (Fig 2f). AdHu5 vector is known as a poor inducer of secreted IL-1\u0026beta;\u003csup\u003e22\u003c/sup\u003e, indicating either lack of induction of pro-IL-1\u0026beta;, or absence of inflammasome activation. BCG can induce transcriptional upregulation of pro-IL-1\u0026beta; but\u0026nbsp;only limited secretion of the active cytokine, in line with its recognised role as a modest activator of the inflammasome pathway\u003csup\u003e23\u003c/sup\u003e. Our data support this, demonstrating secreted IL-1\u0026beta;\u0026nbsp;in the BCG- but not the Ad-vaccinated muscle (Fig 2c, S3).\u003c/p\u003e\n\u003cp\u003eSimilarly, combined Ad+BCG stimulation strongly activated TNF secretion, which is induced by TLR signalling and independent of the inflammasome activation (Fig 2f). Inflammasome-dependent cell death, measured by the release of cytosolic enzyme lactate dehydrogenase (LDH), was found to be vaccine dose-dependent and only elevated at the highest concentrations of Ad+BCG (Fig 2f). These findings demonstrate that, in human macrophages, Ad and BCG strongly synergise in TLR and inflammasome pathway activation which neither of the vaccines can achieve individually. As TLR and inflammasome activation are hallmarks of potent adjuvanticity\u003csup\u003e20\u003c/sup\u003e - a key contributor to effective vaccination - engagement of these pathways is likely to be beneficial for both vaccines.\u003c/p\u003e\n\u003cp\u003eIn summary, we demonstrate that combining BCG with a viral-vectored malaria vaccine generates a broader innate immune response and enables protection against malaria challenge. Simultaneous administration of both vaccines induces a potent inflammatory cytokine profile, capable of supporting protective CD8\u003csup\u003e+\u003c/sup\u003e T cells in this challenge model. Recent studies in mice have shown that intravenously administered BCG can protect against SARS‑CoV‑2 via CD4\u003csup\u003e+\u003c/sup\u003e T cell mediated IFN-\u0026gamma; secretion, through imprinting of myeloid and epithelial cells for enhanced antiviral immunity\u003csup\u003e24,25\u003c/sup\u003e. In clinical trials, previous administration of BCG has been found to enhance the immune response to RNA- or viral vector-based SARS-CoV-2 vaccines\u003csup\u003e26,27\u003c/sup\u003e. Consequently, our findings broaden the portfolio of advantageous applications of the BCG vaccine, identifying co-administration of BCG and malaria vaccines as a potentially promising strategy to combat both TB and malaria in double-endemic regions.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eMice and Immunisations\u003c/h3\u003e\n\u003cp\u003eFor all experiments, mice were purchased from Envigo, UK. Animals were maintained at the Centre for Human Genetics, University of Oxford, housed under Specific Pathogen Free (SPF) conditions in individually ventilated cages, with constant temperature and humidity and a 12 hour light/dark cycle, and in accordance with the recommendations of the UK Animals (Scientific Procedures) Act 1986 and ARRIVE guidelines. Protocols were approved by the University of Oxford Animal Care and Ethical Review Committee for use under Project Licenses P9804B4F1 and PP0984913, granted by the UK Home Office.\u003c/p\u003e\n\u003cp\u003eAnaesthetised (using vaporized IsoFlo\u003csup\u003e\u0026reg;\u003c/sup\u003e) female BALB/c mice (BALB/cOlaHsd) mice, aged 7\u0026ndash;10-weeks, were immunised intramuscularly (i.m.) with a total volume of 50 \u0026mu;L in the tibialis muscle. In the initial experiments BCG was administered intradermally (i.d.) \u0026nbsp;into the ear (2 x 25 \u0026mu;L each site). Comparison with i.m. administration showed no difference in protection against malaria and BCG was given i.m. in subsequent experiments. Each vaccine dose contained 0.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU BCG, or 5 x 10\u003csup\u003e7\u003c/sup\u003e i.u. AdHu5-TIPeGFP.\u003c/p\u003e\n\u003ch3\u003eSporozoite production\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003ePlasmodium berghei\u003c/em\u003e (ANKA strain clone 234) sporozoites were produced as previously described\u003csup\u003e28\u003c/sup\u003e. Starved female \u003cem\u003eAnopheles stephensi\u003c/em\u003e mosquitoes were fed for approximately 10 min on BALB/c mice infected with \u003cem\u003eP. berghei\u003c/em\u003e parasites. The mosquitoes were then maintained on Fructose/PABA solution at 19\u0026ndash;21\u0026deg;C in a humidified incubator on 12 h light/dark cycle. Approximately 21 days after feeding, the mosquitoes were dissected and the salivary glands removed. While keeping on ice, the glands were placed in RPMI-1640 and disrupted with a tissue homogeniser to release the sporozoites. The sporozoites were counted using a haemocytometer, while keeping on ice to preserve viability, and immediately proceeded to malaria challenge.\u003c/p\u003e\n\u003ch3\u003eMalaria challenge\u003c/h3\u003e\n\u003cp\u003eMalaria challenge was performed as described previously\u003csup\u003e28\u003c/sup\u003e. For all experiments 1,000 \u003cem\u003eP.\u0026nbsp;berghei\u003c/em\u003e sporozoites (described above) were injected intravenously in a total volume of 100 \u0026mu;L into the lateral tail vein of each mouse. From day 5 post challenge the mice were monitored for infection by thin-film blood smear (fixed in methanol and stained in 10% Giemsa for 30 min). Mice were sacrificed when \u0026gt;1% parasitaemia was observed. If no parasites were detected on day 12 after challenge, mice were considered sterilely protected.\u003c/p\u003e\n\u003ch3\u003eEx-vivo IFN-\u0026gamma; splenocyte ELISpot\u003c/h3\u003e\n\u003cp\u003eELISpot assays were performed using the IFN-\u0026gamma; ELISpot (kit 3321-2A, Mabtech, UK) as described previously\u003csup\u003e28\u003c/sup\u003e. In brief, two weeks after immunisation mice were culled and spleens removed. Single cell suspensions were prepared by homogenising and straining the spleens followed by brief incubation in ACK lysis buffer to remove erythrocytes. Cells were washed with PBS, pelleted, resuspended in complete \u0026alpha;-MEM and counted using the CASY cell counter. MultiScreen-IP 0.45\u0026thinsp;\u0026mu;m sterile plates were coated with 5\u0026thinsp;\u0026mu;g/ml rat anti-mouse interferon gamma mAb AN18 diluted in carbonate-bicarbonate buffer overnight at 4\u0026thinsp;\u0026deg;C. Plates were then blocked for at least one hour at 37\u0026thinsp;\u0026deg;C with complete \u0026alpha;-MEM. Cells were plated and stimulated for 18\u0026ndash;20\u0026thinsp;h with 1\u0026thinsp;\u0026mu;g/mL of Pb9 peptide. Plates were washed and incubated with 1\u0026thinsp;\u0026mu;g/mL biotinylated anti-mouse-IFN\u0026gamma; mAb R46A2, followed by incubation with 1\u0026thinsp;\u0026mu;g/mL streptavidin alkaline phosphatase. Spots were developed by addition of 50\u0026thinsp;\u0026mu;L per well of colour development buffer and counted using ELISpot software (AID). Results are expressed as spot forming cells (SFC) per million splenocytes after subtracting background responses in unstimulated wells.\u003c/p\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003ePhenotypic analysis of CD8\u003csup\u003e+\u003c/sup\u003e T cells was performed by surface marker staining using the following fluorescently labelled antibodies: PE-Cy7-CD11a (BD Bioscience cat #558191), PE-CD186 (Biolegend cat #151104), BV711-CD69 (Biolegend cat #104537), BV650-CD44 (Biolegend cat #103049), BV605-CD62L (Biolegend cat #104437), BV421-KLRG1 (Biolegend cat #138413), AF700-CD8 (Biolegend cat #100729). Non-specific Ab binding was prevented by incubation with anti-CD16/CD32 Fc\u0026gamma; III/II Receptor (BD Pharmingen, UK) prior to the surface staining. The Pb9 tetramer was provided by the NIH tetramer facility (MHC tetramer core facility, Emory University Vaccine Center, Atlanta, USA) using SYIPSAEKI peptide. Live and dead cells were distinguished using LIVE/DEAD\u003csup\u003eTM\u003c/sup\u003e Fixable Aqua Dead Stain Kit (ThermoFisher Scientific, UK). To define subpopulation distribution within the Pb9\u003csup\u003e+\u003c/sup\u003e compartment (Figure 1d, e) cells were gated on singlets, size, CD8\u003csup\u003e+\u003c/sup\u003e, Pb9\u003csup\u003e+\u003c/sup\u003e, and subsequent definition of subpopulations as depicted in Figure S1d without gating on CD44\u003csup\u003e+\u003c/sup\u003e/CD11a\u003csup\u003e+\u003c/sup\u003e as Pb9\u003csup\u003e+\u003c/sup\u003e cells were generally positive for these markers. Flow cytometric analyses were performed on LSRII (BD Biosciences, UK) and data analysed with FlowJo software.\u003c/p\u003e\n\u003cp\u003eFor B cell profiling, freshly harvested splenocytes were seeded in 96-well plates at a density of ~1.5x10\u003csup\u003e6\u003c/sup\u003e cells per well in chilled FACS buffer. Cells were washed and stained with diluted nIR Zombie L/D stain (Biolegend cat #423105) and incubated for 15 minutes at room temperature. After incubation, cells were washed and stained with a cocktail of the following antibodies diluted in FACS buffer: BV421-CD19 (Biolegend cat #115549), BV605-B220 (Biolegend cat #130244), BV786-CD95 (BD Bioscience cat #740716), PerCPCy5.5-GL7 (Biolegend cat #144609), PECy5-CD38 (Invitrogen cat #15-0381-82), PECy7-CD138 (Biolegend cat #142513), AF700-IgD (Biolegend cat #405729), APCCy7-CD3 (Biolegend cat #100222), APCCy7-F4/80 (Biolegend cat #123117). Cells were subsequently washed and transferred to FACS tubes for flow cytometric analyses on a Fortessa (BD Biosciences, UK).\u003c/p\u003e\n\u003ch3\u003eTotal IgG ELISA\u003c/h3\u003e\n\u003cp\u003eELISAs to detect antibodies against eGFP (expressed by AdHu5-TIPeGFP) or total BCG were performed as previously described\u003csup\u003e28\u003c/sup\u003e. Serum was obtained by collecting blood from the lateral tail vein in a microcuvette tube. Blood was allowed to clot at 4 \u0026deg;C overnight before centrifugation at 13,000\u0026nbsp;rpm for 4\u0026nbsp;min and sera removed and stored at \u0026minus;20 \u0026deg;C until use. For total IgG ELISAs, Nunc-Immuno Maxisorp 96 well plates were coated with 1\u0026nbsp;\u0026mu;g/mL eGFP in carbonate-bicarbonate coating buffer or 10\u003csup\u003e5\u003c/sup\u003e CFU BCG per well overnight at 4 \u0026deg;C. Plates were washed with PBS-Tween (0.05% v/v) and blocked with 2% BSA in PBS-Tween for 1 h at RT. Sera were diluted appropriately between 1:200 and 1:3,200 (depending on Ag and time point) in 1% BSA in PBS-Tween and added to plate in duplicates. Plates were incubated for 2 h at room temperature and then washed as before. Fc-specific goat anti-mouse IgG conjugated to alkaline phosphatase (AP) (1 in 5,000, Sigma-Aldrich, A1418) was added for 1 h at room temperature (eGFP) or overnight at 4\u0026deg;C (BCG). Following a final wash, plates were developed by adding \u003cem\u003ep\u003c/em\u003e-nitrophenylphosphate at 1\u0026nbsp;mg/mL in diethanolamine buffer and OD was read at 405\u0026nbsp;nm. Total IgG concentrations against eGFP or BCG in sera were calculated by interpolation against a standard curve generated from a serum pool of previously AdHu5-TIPeGFP or BCG immunised mice. Sera from naive mice were used as negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA for detection of cytokines in serum, spleen, and muscle (SOI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue cytokines were assessed as described previously\u003csup\u003e29\u003c/sup\u003e. To detect innate cytokines in serum, spleen and muscle (SOI) samples, mice were immunised with AdHu5-TIPeGFP and/or BCG as described above. At specific time points mice were sacrificed, and tissues harvested. Blood samples were allowed to clot for 2 h before centrifugation at 13,000 rpm for 4 min; sera were stored at \u0026minus;80 \u0026deg;C until use. Muscle and spleen samples were weighed, transferred into PBS supplemented with protease inhibitor (Roche, 11873580001), and dissociated in gentleMACS M Tubes using the gentleMACS Octo Dissociator. Cell suspensions were stored at \u0026minus;80\u0026deg;C. Before use, samples were thawed, clarified by centrifugation at 10,000 rpm for 5 min, and supernatants analysed for cytokines and chemokines using the mouse MU Anti-Virus Response Panel flow assay kit (LEGENDplex, Biolegend) according to the manufacturer\u0026rsquo;s instructions. LSR Fortessa (BD) flow cytometer was used to assess fluorescence intensity of beads and data analysed using LEGENDplex Data Analysis Software Suite.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Monocyte Derived Macrophages (HMDM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAssays on Human Monocyte Derived Macrophages were performed as previously described\u003csup\u003e29\u003c/sup\u003e. In brief, peripheral blood mononuclear cells were isolated using Ficoll gradient from healthy donors from NHS Oxford blood bank (REC approval 11/H0711/7). Magnetic beads (eBioscience, 8802-6834-74) were used to positively select CD14\u003csup\u003e+\u0026nbsp;\u003c/sup\u003emonocytes which were differentiated into macrophages by culturing for 7\u0026nbsp;days with M-CSF (100\u0026nbsp;ng/mL, BioLegend, 574808). Cells were cultured in RPMI (Fisher, 21870076) with 10% FBS (Life Technologies, 16000044) and 1x Pen/Strep/Glutamine (Fisher, 10378016) at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, and supplemented with fresh media containing 100\u0026nbsp;ng/mL M-CSF on day 2 and day 5. After day 7 of differentiation, cells were re-plated (70,000 cells per well in flat bottom 96-well plates in 100\u0026nbsp;\u0026mu;L complete RPMI) and on day 8 cells were stimulated for inflammasome-activation experiments.\u003c/p\u003e\n\u003cp\u003eDifferentiated HMDMs were plated at a density of 0.7x10\u003csup\u003e6\u003c/sup\u003e cells/mL complete RPMI and stimulated for 24 h with AdHu5-TIPeGFP and/or BCG at concentrations indicated in the Figures. Culture media was replaced with 100 \u0026micro;L OptiMEM (Fisher, 31985062) immediately before stimulation. After cell stimulation, cell supernatants were collected for analysis. Secretion of IL-1\u0026beta; and TNF was measured in cell-free supernatants using ELISA (Thermo Fisher, 88-7261-77 and 88-7346-88) according to manufacturer\u0026rsquo;s instructions. Cellular viability was assessed using cell culture supernatants and the Cytox96 nonradioactive cytotoxicity assay (Promega, G1780).\u003c/p\u003e\n\u003ch3\u003eStatistical analysis\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was performed using Prism software (V10). Statistical tests applied and pairwise comparisons with correction for multiple testing are indicated in the Figure legends. In all experiments\u0026nbsp;\u003csup\u003e\u0026lowast;\u003c/sup\u003ep\u0026nbsp;\u0026lt;\u0026nbsp;0.05,\u0026nbsp;\u003csup\u003e\u0026lowast;\u0026lowast;\u003c/sup\u003ep\u0026nbsp;\u0026lt;\u0026nbsp;0.01,\u0026nbsp;\u003csup\u003e\u0026lowast;\u0026lowast;\u0026lowast;\u003c/sup\u003ep\u0026nbsp;\u0026lt;\u0026nbsp;0.001,\u0026nbsp;\u003csup\u003e\u0026lowast;\u0026lowast;\u0026lowast;\u0026lowast;\u003c/sup\u003ep \u0026lt; 0.0001. Non-significant differences (n.s.) are generally not indicated.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis work was funded in part by the MRC Confidence in Concept Award MC_PC_16056 (A.M.); VaxHub Global grant, Department of Health and Social Care using UK Aid funding, with support from EPSRC as part of the UK Vaccine Network (UKVN), a UK Aid programme to develop vaccines for diseases with epidemic potential in low and middle-income countries (LMICs) (A.M); Chan Zuckerberg Initiative grant 2020-217289 (A.M., J.F.), UK Medical Research Council MR/Y004450/1 (J.F.); NIHR Biomedical Research Centre, Inflammation Across Tissues Theme (J.F.); the Kennedy Trust for Rheumatology Research KENN212202 (J.S.B.), Medical Research Council MR/W001217/1 (J.S.B.); and Kennedy Trust for Rheumatology Research Studentship (S.Q.), The Clarendon Fund in partnership with an Oxford-St Cross E.P. Abraham Scholarship and Nuffield Department of Clinical Medicine Studentship (L.S.), Michelson Prize 2022 (R.G.). S.R. was funded in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 495054088).\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank Elena Stylianou for sharing the expertise on growing BCG and intradermal injections, and Mariya Mykhaylyk for lab-keeping assistance. H2-Kd\u003cem\u003e\u0026nbsp;P. berghei\u003c/em\u003e CSP 245-253 SYIPSAEKI tetramer was kindly provided by the NIH Tetramer Core Facility (contract number 75N93020D00005). Illustrations were created with BioRender.com.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution (CRediT)\u003c/h2\u003e\n\u003cp\u003eConceptualization: JRF, AM\u003c/p\u003e\n\u003cp\u003eMethodology: SR, SQ, LS, JSB, AM\u003c/p\u003e\n\u003cp\u003eValidation: SR, JRF, AM\u003c/p\u003e\n\u003cp\u003eFormal analysis: SR, RG, JRF \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigation: SR, MU, SQ, LS, RG, AT, JRF, AM\u003c/p\u003e\n\u003cp\u003eResources:\u0026nbsp;JSB, AM\u003c/p\u003e\n\u003cp\u003eData Curation: SR, JRF, AM\u003c/p\u003e\n\u003cp\u003eWriting - Original Draft:\u0026nbsp;SR\u003c/p\u003e\n\u003cp\u003eWriting - Review \u0026amp; Editing: SR, MU, SQ, LS, RG, JSB, JRF, AM\u003c/p\u003e\n\u003cp\u003eVisualization: SR\u003c/p\u003e\n\u003cp\u003eSupervision:\u0026nbsp;AM\u003c/p\u003e\n\u003cp\u003eProject administration: JSB, JRF, AM\u003c/p\u003e\n\u003cp\u003eFunding acquisition: SR, JSB, AM\u003c/p\u003e\n\u003ch2\u003eDeclaration of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJurczak, M. \u0026amp; Druszczynska, M. 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This may stem from its ability to epigenetically imprint innate immune cells, enhancing subsequent response to pro-inflammatory stimuli, coupled with intrinsic adjuvanticity through direct activation of cell surface PRRs. Here we investigate whether vaccinating with BCG in parallel with an adenoviral-vectored vaccine could be beneficial for protection against malaria in a mouse model. We show that combining the two vaccines broadens and strengthens the early innate cytokine repertoire, enabling strong CD8\u003csup\u003e+\u003c/sup\u003e T cell-dependent protection. Our findings reveal co-immunisation with BCG and a viral vector as a promising strategy to combat both TB and malaria in double-endemic regions.\u003c/p\u003e","manuscriptTitle":"BCG enables protection against malaria through adjuvanting an Adenovirus-vectored vaccine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 08:14:45","doi":"10.21203/rs.3.rs-7858925/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-07T18:12:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T12:24:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236448019777544036987361397589223720954","date":"2025-11-22T14:58:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-15T13:02:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212499532207463341115462662723263077247","date":"2025-11-10T04:28:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-08T03:55:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T17:05:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T11:30:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-10-14T12:55:50+00:00","index":"","fulltext":""}],"status":"published","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}}],"origin":"","ownerIdentity":"b06f2e90-eb14-4d1f-8286-b4ed27e12d39","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56635141,"name":"Health sciences/Diseases"},{"id":56635142,"name":"Biological sciences/Immunology"},{"id":56635143,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-17T11:24:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-22 08:14:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7858925","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7858925","identity":"rs-7858925","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}