{"paper_id":"355a2cca-dae3-42e8-ab15-347d2cfeadc1","body_text":"A viral vaccine design harnessing prior BCG immunization confers 1 \nprotection against Ebola virus 2 \nTony W. Ng1, Wakako Furuyama2, Ariel S. Wirchnianski1, Noemí A. Saavedra-Ávila1, 3 \nChristopher T. Johndrow1, Kartik Chandran1, William R. Jacobs, Jr.1, Andrea Marzi2, Steven 4 \nA. Porcelli1,3 5 \n1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 6 \nUSA 7 \n2Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and 8 \nInfectious Diseases, National Institute of Health, Hamilton, MT, USA 9 \n3Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA 10 \n* Correspondence:  11 \nSteven A. Porcelli 12 \nsteven.porcelli@einsteinmed.edu 13 \nKeywords: Ebola virus, Mycobacterium bovis BCG, CD4+ T cells, antibodies, vaccines, 14 \nintrastructural help, Fc receptors. 15 \nAbstract 16 \nPrevious studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that 17 \ntakes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille 18 \nCalmette-Guérin (BCG) vaccination.  This strategy uses immunization with recombinant fusion 19 \nproteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, 20 \nengineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by 21 \nprior BCG vaccination to provide intrastructural help to virus-specific B cells.  In the current study, 22 \nwe show that Th cells induced by BCG were localized predominantly outside of germinal centers and 23 \npromoted antibody class switching to isotypes characterized by strong Fc receptor interactions and 24 \neffector functions.  Furthermore, BCG vaccination also upregulated FcR expression to potentially 25 \nmaximize antibody-dependent effector activities.  Using a mouse model of Ebola virus (EBOV) 26 \ninfection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and 27 \nprotection against lethal challenge.  This general approach can be easily adapted to other viruses, and 28 \nmay be a rapid and effective method of immunization against emerging pandemics in populations 29 \nthat routinely receive BCG vaccination. 30 \n1 Introduction 31 \nEmergent viruses such as Ebola virus (EBOV) and related filoviruses are global health threats of 32 \nincreasing concern, especially due to the expansion of human populations into wild habitats that 33 \nserve as natural reservoirs for these viruses 1.  For prevention of outbreaks of viral infections or 34 \npandemics, vaccines remain the most practical and cost-effective tools.  This has clearly been shown 35 \nin the ongoing Coronavirus disease 2019 (COVID-19) pandemic where vaccination has been 36 \nreported to reduce the risk of severe illness leading to hospitalization and mortality rates among 37 \nvaccinated individuals 2.  Historically, vaccine development has been mainly focused on the variable 38 \n(Fab) region of immunoglobulins for their ability to bind surface antigens of viruses and prevent 39 \nentry into host cells 3-5.  Such neutralizing antibodies (NAbs) are a major correlate of protection 40 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 2 \nassociated with viral clearance and resolution of the infection.  However, the limited range of 41 \nepitopes available to induce NAbs may prevent efficient clearance of infection by this mechanism for 42 \nsome viruses.  Furthermore, as shown in human immunodeficiency virus (HIV) infection, NAbs exert 43 \nstrong selective pressure in driving immune escape of the virus as compared to non-neutralizing 44 \nantibodies 6.  These observations suggest that eliciting non-neutralizing antibodies mediating effector 45 \nfunctions distinct from simple blockade of viral entry might increase the protective efficacy of a 46 \nvaccine 7,8.   47 \nThe constant (Fc) regions of antibodies, although recognized as important in contributing to 48 \nprotection, have been less emphasized compared to the Fab region as a determinant of anti-viral 49 \neffects.  The Fc region binds to the Fc receptors (FcRs) on a variety of relevant immune effector 50 \ncells, thus bridging humoral and cellular immunity through effector activities such as antibody-51 \ndependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-52 \ndependent cellular cytotoxicity (ADCC) that are believed to contribute to control of viral and other 53 \nmicrobial infections 9,10.  Recently, results from clinical trials for newly developed EBOV and HIV 54 \nvaccines have called attention to the importance of antibody-mediated effector functions as correlates 55 \nof protection against viral pathogens 11-15.  This has driven the search for relevant antibody effector 56 \nfunctions beyond simple neutralization in individuals vaccinated against or exposed to EBOV 16,17 or 57 \nHIV 18,19, and has encouraged efforts to engineer therapeutic antibodies with optimal Fc effector 58 \nfunctions for these diseases 20,21. 59 \nWhereas the binding affinity of the Fab region of the antibody develops and matures in the 60 \ngerminal centers (GC) within B cell follicles of secondary lymphoid tissues 22, class-switch 61 \nrecombination (CSR) required for determining Fc isotype is initiated and occurs mostly at the border 62 \nof the B cell follicle between the boundary of B and T cell zones 23.  Activation of  CSR requires 63 \nsignals from B cell receptor (BCR) engagement, costimulatory signals such as CD40-CD40L 64 \ninteraction, and particularly cytokines secreted from CD4+ helper T cells (Th) that dictate which 65 \nswitch region of the heavy chain constant region genes will interact with activation-induced cytidine 66 \ndeaminase (AID) to initiate the double strand DNA break required for recombination to occur 24.  67 \nTherefore, the ability to induce different Th phenotypes, such as Th1, Th2 or follicular helper T cells 68 \n(Tfh), during vaccination can have an impact on class-switching of immunoglobulins.  In C57BL/6 69 \nmice, for example, class-switching to IgG2c (homologous to IgG2a in other mouse strains 25), is 70 \ninduced by IFN produced by Th1 cells 26,27, whereas IgG1 is induced by IL-4 derived mainly from 71 \nTh2 cells  28-30.  These antibody subclasses have different affinities to particular Fc receptors (FcRs) 72 \n31.  In mice, antibodies with the IgG1 isotype have low, but similar affinities for the inhibitory 73 \nFcRIIB and the activating FcRIII, whereas the affinities conferred by the IgG2 isotypes for the 74 \nactivating  FcRIV are much stronger 31.  Under inflammatory Th1 conditions, IgG2c class switching 75 \nand an increased expression of FcRIV are favored 32, thus promoting effector functions such as 76 \nphagocytosis, complement activation and cytotoxicity, all contributing to the removal of either the 77 \npathogen itself or the cells infected by it. 78 \nMycobacterium bovis bacille Calmette-Guérin (BCG), the only currently approved vaccine 79 \nagainst tuberculosis, is one of the most widely administered vaccines in many regions of the world.  80 \nThe BCG vaccine induces long lasting BCG-specific memory CD4+ T helper cells (Th) that are 81 \nstrongly polarized to IFNγ-secreting Th1 cells in vaccinated individuals.  To take advantage of the 82 \nhigh prevalence of BCG vaccination, we developed a vaccination strategy that uses pre-existing 83 \nBCG-specific Th cells to drive antibody responses against a modified viral protein immunogen 33.  84 \nThis recombinant fusion protein vaccine (Th-vaccine), based on a principle previously designated 85 \nintrastructural help 34,35, induces antiviral antibody responses with a strong bias to IgG2c isotype in 86 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n3 \nC57BL/6 mice 33.  In the current study, we have used an EBOV challenge model to show that this 87 \napproach promotes antibodies that can recruit effector cells capable of eliminating virus infected cells 88 \nand is highly effective at protecting mice from lethal virus challenge. Analysis of the underlying 89 \nmechanism for the effects on the antibody response showed that BCG vaccination created 90 \ninflammatory conditions that impaired GC formation, similar to what has been seen in infections 91 \nwith other Th1 skewing pathogens 36-39.  Antibody class switching thus occurred outside of the GC, 92 \nleading to a strong bias of anti-EBOV antibodies to IgG2c isotype due to the influence of BCG-93 \nspecific Th1 cells.  The anti-EBOV GP antibodies elicited by this vaccination regimen were 94 \nmaintained over time suggesting the induction of long-term protection.  Overall, our findings support 95 \nthe importance of non-neutralizing antibodies in anti-viral vaccination, and define a powerful and 96 \npotentially useful method to induce such antibodies against established or newly emerging viruses in 97 \npopulations that receive routine BCG vaccinations. 98 \n2 Materials and Methods 99 \nMice 100 \n 101 \nFive-week old female wild-type (WT) C57BL/6NHsd and C57BL/6J mice were obtained from 102 \nEnvigo (Greenfield, IN) and The Jackson Laboratory (Bar Harbor, ME), respectively.  The GFP+ 103 \nC57BL/6-P25 TCR transgenic (Tg) mice 33 with T cell receptor that recognizes the P25 peptide 104 \n(FQDAYNAGGHNAVF) from M. tuberculosis or BCG Ag85B were maintained and bred in our 105 \nfacility.  All mice were maintained in our specific pathogen-free facilities following protocols and 106 \nregulations established by the Albert Einstein College of Medicine Institutional Animal Use and Care 107 \nand the Institutional Biosafety Committees.  All procedures performed on these animals were 108 \napproved by the Albert Einstein College of Medicine Institutional Animal Use and Care Committee. 109 \n 110 \n 111 \nMycobacterial strains and vaccinations 112 \n 113 \nMycobacterium bovis BCG Danish strain (Statens Serum Institut, Copenhagen, Denmark) was the 114 \nBCG vaccine strain used in this study.  Starting from a low-passage-number frozen stock, the 115 \nbacteria was grown at 37C shaking in Sauton medium until mid-log phase, centrifuged at 600 x g for 116 \n10 minutes, and resuspended in sterile PBS (Thermo Fisher Scientific, Waltham, MA).  BCG was 117 \nadministered by subcutaneous (s.c.) injection at the scruff of the neck at a dose of 1 x 107 CFU.  For 118 \nrecombinant protein vaccines injections, the vaccine in PBS was mixed in a 1:1 volume ratio with 119 \nalum suspension (Imject Alum; Thermo Fisher Scientific) to a final concentration of 0.5 µg/ml unless 120 \notherwise specified, and 100 µl was administered intramuscular (i.m.) into the thigh muscles with 50 121 \nµl per hind limb to provide the final dose of 0.05 µg of the recombinant protein vaccine per animal. 122 \n 123 \n 124 \nCell lines 125 \n 126 \nFreeStyle 293-F cells (Thermo Fisher Scientific) were maintained in Life Technologies FreeStyle 127 \n293 Expression Medium with GlutaMAX (Thermo Fisher Scientific).  Murine T cell hybridomas 128 \n(TCHs) specific for I-Ab -restricted CD4 T cell epitopes 33 were maintained in complete RPMI 129 \n(cRPMI) which consists of RPMI 1640 (Thermo Fisher Scientifics) supplemented with 10 mM 130 \nHEPES, 50 µg/ml penicillin/streptomycin, 55 µM 2-mercaptoethanol (Thermo Fisher Scientifics), 131 \nand 10% heat-inactivated [56ºC, 30 min] fetal bovine serum (Atlanta Biologicals, Flowery Branch, 132 \nGA). 133 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 4 \n 134 \n 135 \nPlasmid construction 136 \n 137 \nThe recombinant protein vaccine that consists of the extracellular portions of EBOV GP lacking the 138 \nMLD (WT GPΔM) and a similar version that consists of BCG Th epitopes fused to the N terminus of 139 \nthe EBOV GP (Th GPΔM) were constructed and described previously 33.  The full-length EBOV GP 140 \nwith the MLD restored was also constructed for these recombinant protein vaccines.  To construct the 141 \nfull-length version of the EBOV GP vaccines, the MLD of the EBOV GP was amplified from 142 \npMAM01 40 using primer pair TN258 (5΄-143 \nGCGCACCGTCGTGTCAAACGGAGCCAAAAACATCAGTGG-3΄) and TN259 (5΄-144 \nGCGCCAGTATCCTGGTGGTGAGTGTTGTTGTTGCCAGCGG-3΄).  The MLD was cloned into 145 \nWT GP-ΔM and Th GP ΔM via the AleI and XcmI sites to create the corresponding version WT GP-146 \nFL and Th GP-FL which contain the MLD in the EBOV GP. 147 \n 148 \n 149 \nExpression and purification of recombinant protein vaccines 150 \n 151 \nPlasmids corresponding to WT GP-FL and Th GP-FL DNA were transfected into FreeStyle 293-F 152 \ncells, and proteins were collected from culture supernatants and purified using the HisTrap HP 153 \ncolumn (GE Healthcare Life Sciences, Pittsburgh, PA) as described previously 33.  Protein 154 \nconcentrations were determined by the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).  155 \nThe purified proteins (220ug/ml WT GP-GL and 80ug/ml Th GP-FL) in PBS were stored at -80ºC 156 \nuntil needed. Purified Th vaccines were analyzed by size-exclusion high performance liquid 157 \nchromatography (SE-HPLC) using the SRT SEC-300 size exclusion column.  Analysis of the 158 \nmolecular weight was determined by comparing the retention time with markers of known molecular 159 \nweight (BioRad). 160 \n 161 \n 162 \nSDS-PAGE analysis of recombinant protein vaccines 163 \n 164 \nPurified recombinant protein vaccines were analyzed on SDS-PAGE by staining with GelCode Blue 165 \nSafe Protein Stain (Thermo Fisher Scientific).  Proteins separated by SDS-PAGE were also 166 \ntransferred onto nitrocellulose membranes for immunoblotting.  After blocking with 5% bovine milk 167 \nin PBS with 0.05% Tween 20 (PBST), the nitrocellulose membranes containing the purified 168 \nrecombinant fusion proteins were incubated with mouse anti-His antibody [clone HIS.H8] (Millipore 169 \nSigma, Burlington, Massachusetts).  HRP-conjugated rabbit anti-mouse IgG antibody 170 \n(SouthernBiotech, Birmingham, AL) were used as detection Abs, and signals were detected using the 171 \nSuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). 172 \n 173 \n 174 \nT cell hybridoma stimulation assays 175 \n 176 \nMouse T cell hybridomas specific for peptide P25 of Ag85 or peptide P10 of TB9.8 were cocultured 177 \nwith murine bone marrow-derived dendritic cells 33 and incubated with the purified recombinant 178 \nprotein vaccine (10 µg/ml) at 37ºC for 18 h.  Cell culture supernatants were assayed for IL-2 using 179 \ncapture and biotin-labeled detection antibody pairs (BD Biosciences, Franklin Lakes, NJ).  Detection 180 \nwas performed with HRP-conjugated streptavidin (BD Biosciences) followed by the addition of the 181 \nTurbo 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific). 182 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n5 \n 183 \n 184 \nELISA assays for anti-EBOV GP antibody titers 185 \n 186 \nMeasurement of anti-EBOV GP antibody titers was performed by direct solid phase ELISA.  Corning 187 \n96-well flat bottom assay plate (Thermo Fisher Scientific) were coated with ~95,000 infectious unit 188 \n(IU) of recombinant vesicular stomatitis virus (rVSV) expressing EBOV GP (rVSV-EBOV) in PBS 189 \n(pH7.4) overnight at 4ºC 41,42.  The EBOV GP coated ELISA plate was washed three times with PBS 190 \nand blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at RT.  Serum samples from 191 \nimmunized mice were obtained from blood collected by retro-orbital bleed in vaccinated mice.  The 192 \nserum samples were diluted 1:50 in PBS for single dilution measurement or 1:20 followed by serial 193 \n1:3 dilutions for endpoint titers, and then incubated in the EBOV GP coated ELISA plate wells for 2 194 \nh at RT.  The ELISA plates were then washed four times with PBS and incubated with HRP-195 \nconjugated mouse IgG1- or IgG2c-specific Abs (SouthernBiotech) for 1 h at RT.  After washing four 196 \ntimes in PBS, the signal was detected with SIGMAFAST OPD substrate (Sigma-Aldrich, St. Louis, 197 \nMO) and the reaction was stopped with the addition of 0.5 M H2SO4.  The absorbances for both the 198 \ncapture and direct ELSIA assays were measured with the Wallac 1420 VICTOR2 microplate reader 199 \n(Perkin Elmer, Waltham, MA).      200 \n 201 \n 202 \nFlow cytometry analysis of Th subsets and FcR expression 203 \n 204 \nNaïve CD4+ T cells from P25 TCR-Tg/GFP mice were purified by negative selection using a 205 \ncommercially available kit and following the manufacturer’s instruction (Miltenyi Biotec, Auburn, 206 \nCA).  During the CD4+ T cell purification, anti-CD44 conjugated to biotin [clone IM7] (Thermo 207 \nFisher Scientific) was added in the purification step to remove memory T cells that were present in 208 \nthese animals.  4 x 104 purified CD4+ T cells in 100 µl of PBS were injected intravenously via the tail 209 \nvein into WT C57BL/6 mice.  Sixteen hours after injection of the CD4+ T cells, mice were vaccinated 210 \nwith 100 µl of either 1 x 107 CFU of BCG in PBS or with 10 µg P25 peptide in PBS formulated with 211 \none of the following adjuvants: 1:1 volume ratio of alum (Imject Alum; Thermo Fisher Scientific), or 212 \n5% final volume of LASTS-C [Span85-Tween 80-squalene, lipid A, CpG oligodeoxynucleotides] 213 \n43,44 (gift from Dr. Michael Anthony Moody, Duke University).  On day 7 after vaccination, mice 214 \nwere sacrificed, and spleens were harvested and cells were stained with Live Dead viability dye (LD 215 \nFixable Blue; Thermo Fisher Scientific L34961) and antibodies against MHC class II (Alexa Fluor 216 \n700; BD 570802), CD4 (APC-Cy7, BD 561830), T-bet (PE-Cy7; Biolegend 644823), CXCR-5 (PE; 217 \nBD 551959), and Bcl-6 (APC; Biolegend 358505), and analyzed by FACS using the 5 laser BD 218 \nBiosciences LSRII Flow Cytometer, and 5 x 105 events per sample were collected and analyzed using 219 \nFlowJo software (BD biosciences). 220 \nFor analysis of FcR expression, splenocytes from FcRII,III,IV -chains knockout mice 45 or 221 \nWT B6 vaccinated mice were processed at indicated timepoints and stained with mAbs against B220 222 \n(BUV661; BD 612972, clone: RA3-6B2), NK1.1 (BV605; BD 563220, clone: PK136), CD11c 223 \n(Alexa Fluor 700; BD 560583, clone: HL3), CD11b (PE-CF594; BD562287, clone: M1/70), Ly-224 \n6G/Ly-6C (APC; eBioscience 17-5931-81, clone: RB6-8C5), Ly6-C (PerCP; Biolegend 128028. 225 \nclone: HK1.4), FcRIV (PE; BD 565615, clone: 9E9), FcRII/III (FITC; BD 561726, clone: 2.4G2), 226 \nand analyzed by FACS using the Cytek Aurora configured with five lasers, three scattering channels, 227 \nand sixty-four fluorescence channels, and 1 x 106 events per sample were collected and analyzed 228 \nusing the FlowJo software (BD biosciences). 229 \n 230 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 6 \n 231 \nAnalysis of germinal centers 232 \n 233 \nTo detect the presence of antigen specific Th cells in the secondary lymphoid tissues, spleens from 234 \nvaccinated mice were sectioned and stained as described previously 33.  Briefly, naïve CD4+ T cells 235 \nfrom P25 TCR-Tg/GFP mice were transferred into mice which were then vaccinated with either 1 x 236 \n107 BCG or with 10 µg P25 peptide formulated in LASTS-C as described in the analysis of Th 237 \nsubsets above.  On day 7 after vaccination, mice were sacrificed and spleens were fixed in 10% 238 \nneutral buffered formalin, paraffin embedded and sectioned.  Tissue sections were stained with anti-239 \nGFP Ab (A11122; Thermo Fisher Scientific) for the presence of CD4+ T cells transferred from the 240 \nP25 TCR-Tg/GFP mouse and counterstained with hematoxylin. 241 \n 242 \n 243 \nAnimal ethics statement 244 \n 245 \nAll infectious work with MA-EBOV was performed in the maximum containment laboratories at the 246 \nRocky Mountain Laboratories (RML), Division of Intramural Research, National Institute of Allergy 247 \nand Infectious Diseases, National Institutes of Health. RML is an institution accredited by the 248 \nAssociation for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 249 \nAll procedures followed standard operating procedures (SOPs) approved by the RML Institutional 250 \nBiosafety Committee (IBC). Mouse work was performed in strict accordance with the 251 \nrecommendations described in the Guide for the Care and Use of Laboratory Animals of the National 252 \nInstitute of Health, the Office of Animal Welfare and the Animal Welfare Act, United States 253 \nDepartment of Agriculture. The study was approved by the RML Animal Care and Use Committee 254 \n(ACUC). Procedures were conducted in mice anesthetized by trained personnel under the supervision 255 \nof veterinary staff. All efforts were made to ameliorate animal welfare and minimize animal 256 \nsuffering; food and water were available ad libitum. 257 \n 258 \n 259 \nEBOV challenge in vaccinated mice 260 \n 261 \nWild-type female C57BL/6NHsd (approximately 10 weeks of age) were given 1 x 107 BCG in PBS 262 \nthrough s.c. injection at the scruff of the neck.  Five weeks after exposure to BCG, the mice were 263 \nprimed with 0.05 µg of the purified recombinant protein vaccine (WT GP-FL or Th GP-FL) 264 \nadjuvanted with alum in PBS through i.m. injection as described above.  Vaccinated mice were rested 265 \nfor 4 weeks, followed by a homologous boost of the recombinant protein vaccine administered 266 \nthrough the same route.  Two weeks after each interval of administering the purified recombinant 267 \nprotein vaccine, blood was collected through retro-orbital bleed to obtain serum samples for antibody 268 \ntiter measurements.  Four weeks after the boost, mice were shipped to Rocky Mountain Laboratories 269 \nin Hamilton, MT and rested for 1 week prior to MA-EBOV challenge.  Mice were infected by 270 \nintraperitoneal (i.p.) injection of a lethal dose for naïve mice of 10 focus-forming units (FFU) of MA-271 \nEBOV 46.  Five mice from each vaccinated group were euthanized on day 5 after challenge to harvest 272 \norgans to determine viremia and to collect blood samples to freeze down serum samples for future 273 \nanalysis of anti-EBOV GP antibody responses.  The remaining 10 mice from each vaccinated group 274 \nwere kept under observation for survival and weight loss and all surviving mice were euthanized on 275 \nday 28 after challenge to collect and freeze serum samples. 276 \n 277 \n 278 \nELISPOT to detect antigen specific T and B cells 279 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n7 \n 280 \nFor the T cell analysis, five-week-old female WT C57BL/6J mice (n = 10) were vaccinated by s.c. 281 \ninjection of either PBS or BCG and rested for 5 weeks.  Each group was further subdivided into 2 282 \ngroups (n = 5) which received either PBS or the Th GP-FL vaccine at 0.05 μg per mouse in alum.  283 \nSplenocytes were obtained two weeks later for ELISPOT assay 47.  Briefly, the 96-well ELISPOT 284 \nplate (Millipore) was prepared by coating the well with 50 μl of 10 μg/ml of anti-mouse IFN 285 \nmonoclonal capture antibody (BD Biosciences; cat. no. 551309) in PBS and allowed to incubate at 286 \n4ºC for 16 hours.  The ELISPOT wells were washed five times with PBST and blocked with 200 μg 287 \nof cRPMI for 2 hours at room temperature.  Splenocytes at 5 x 105 cells per well were added along 288 \nwith 5 μg/ml P25 peptide or 10 μg/ml of M. tuberculosis (strain H37Rv) lysate for antigen 289 \nstimulation and incubated at 37ºC in 5% CO2 for 16 hours.  The ELISPOT plate was washed five 290 \ntimes with PBST and 50 μl of 1 μg/ml of the anti-mouse IFN monoclonal detection antibody 291 \nconjugated to biotin (BD Biosciences; cat. no. 551506) in PBS was added and allowed to incubate at 292 \nroom temperature for 2 hours.  The wells were then washed five times with PBS + 0.1% Tween-20 293 \n(PBST) and streptavidin-alkaline phosphatase (Thermo Fisher Scientific) at 1:1000 dilution in PBS 294 \nwas added incubated at 37ºC in 5% CO2 for 1 hour.  After a final 5 washes with PBST, the spots 295 \nwere developed by adding the BCIP/NBT substrate (Sigma Aldrich).  The reaction was stopped by 296 \nwashing the wells with water and the spots were counted using an automated ELISPOT reader 297 \n(Autoimmun Diagnostika GmbH, Strasbourg, Germany).  298 \nFor ELISPOT quantitation of antibody secreting cells 48, five-week-old female WT C57BL/6 mice 299 \nwere vaccinated with PBS or BCG by s.c. administration of 1 x 107 CFU per mouse.  Five weeks 300 \nafter vaccination, mice were injected i.m. with 5 μg of the Th GP-FL vaccine adjuvanted with alum 301 \nin PBS.  Thirty-nine weeks later, the mice were sacrificed to obtain the splenocytes and bone marrow 302 \ncells, which were immediately assayed by ELISPOT to detect antibody secreting B cells.  The 96-303 \nwell ELISPOT plate (Millipore) was prepared by coating the wells with 50 μl of 10 μg/ml of rVSV 304 \nexpressing EBOV GP and incubating at 4ºC for 16 hours.  The ELISPOT wells were washed five 305 \ntimes with PBS and blocked with 200 μl of cRPMI for 2 hours at room temperature.  Splenocytes or 306 \nbone marrow cells at 106 cells per well were added to the ELISPOT plate and incubated at 37ºC in 307 \n5% CO2 for 5 hours.  The ELISPOT plate was washed five times with PBS and anti-mouse IgG1 or 308 \nanti-mouse IgG2c antibodies conjugated with alkaline phosphatase (Southern Biotech) at 1:1000 in 309 \nPBS were added and incubated at room temperature for 2 hours.  The spots were developed by 310 \nadding the BCIP/NBT substrate (Sigma Aldrich).  The reaction was stopped by washing the wells 311 \nwith water and the spots were counted using an automated ELISPOT reader (Autoimmun 312 \nDiagnostika GmbH, Strasbourg, Germany). 313 \nComparative immunogenicity of EBOV GP vaccine constructs 314 \n 315 \nSubunit vaccines against EBOV have shown potential for inducing protection against infection in 316 \nseveral animal models and may have important advantages over virally vectored vaccines 49.  317 \nHowever, optimal design of subunit vaccines regarding efficacy, potency, stability and formulation 318 \nissues requires further investigation and testing 50.  The design of the EBOV glycoprotein (GP) Th-319 \nvaccine for the current study was based on our previous work showing the general impact of 320 \nincorporating immunodominant Th epitopes of BCG into a soluble version of EBOV GP from which 321 \nthe mucin-like domain (MLD; EBOV GPMLD) was deleted to direct responses against conserved 322 \nepitopes important for neutralizing antibodies 33,40.  Here we developed a new version of the EBOV 323 \nGP Th-vaccine that consisted of the full-length complete extracellular portion of the EBOV GP for 324 \ndirect comparison with the previous version of EBOV GPMLD.  Although the EBOV GPMLD 325 \nwas produced with higher yields as a recombinant protein, the full-length version of the EBOV GP 326 \nTh-vaccine has the advantages of more closely resembling the native protein on the viral envelope or 327 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 8 \nsurface of infected cells, and also could provide a greater range of epitopes for antibody targeting of 328 \nmembrane expressed GP.  As shown schematically (Fig. 1A), the immunodominant CD4+ T cell 329 \nepitopes of mycobacterial antigens Ag85B (P25 epitope) and TB9.8 (P10 epitope) were fused to the 330 \nN terminus of the full-length EBOV GP or to a version of EBOV GPMLD.  These were designated 331 \nTh GP-FL or Th GP-ΔM, respectively.  Protein expression was done in FreeStyle 293-F cells and 332 \npurified by Ni-NTA affinity chromatography as described previously 33.  Versions of these proteins 333 \nlacking the N-terminal extension encoding the BCG Th epitopes, designated as wild type (WT), were 334 \nalso constructed and purified to serve as controls.   335 \nProtein purity and quality were assessed by SDS-PAGE and immunoblotting.  As shown by 336 \nCoomassie staining (Fig. 1B, left panel), the mature form (GP0) and the two proteolytic fragments 337 \n(GP1 and GP2) of the full-length (WT GP-FL and Th GP-FL) and the MLD versions (WT GP-ΔM 338 \nand Th GP-ΔM) of EBOV GP constructs were observed and confirmed to be of the expected sizes 339 \n40,51.  As expected, immunoblotting with antibody specific for the hexahistidine tag at the carboxyl-340 \nterminal of the 25 kDa GP2 precursor (Fig. 1B, right panel) detected the mature form GP0 and the 341 \nGP2 cleavage product, but not the GP1 fragment which lacks the histidine tag.  Since EBOV GP 342 \nexists mainly as trimers in its native cell surface form, we also carried out size exclusion 343 \nchromatography to analyze monomeric versus multimeric state of the soluble GP constructs in 344 \nsolution 52,53 (Supplemental Fig. 1).  This showed retention times consistent with mass of 600 kDa or 345 \nmore for the proteins in solution, indicating complexes at least as large or larger than the expected 346 \nsize for soluble trimers.  This suggested that the subunit vaccines produced here were likely to be a 347 \nmixture of trimers and higher order multimers. 348 \nConsistent with the correctly folded structure for at least a fraction of the purified GP 349 \npreparations, the conformation sensitive anti-EBOV GP antibodies ADI-15878 and KZ52 54 bound to 350 \nall of the purified proteins in solid phase ELISA, (Fig. 1C and Supplemental Fig. 2).  To demonstrate 351 \ncorrect processing and presentation of the BCG epitopes embedded in the Th (FL) and Th (ΔM) 352 \nfusion proteins for T cell recognition, we used previously isolated mouse T cell hybridomas specific 353 \nfor the Ag85B or TB9.8 epitopes presented by MHC class II I-Ab molecules 33,55.  T cell hybridoma 354 \ncells cultured with mouse bone marrow derived dendritic cells secreted IL-2 into the culture 355 \nsupernatants in response to the purified GPs containing the Th sequence encoding the relevant T cell 356 \nepitopes, indicating efficient antigen processing at the inserted cathepsin S cleavage sites and 357 \npresentation by I-Ab (Fig. 1D).  Furthermore, the BCG epitopes incorporated into the Th vaccines 358 \nwere targeted by long-lived memory Th cells in BCG vaccinated mice.  This was apparent in mice 359 \nvaccinated with PBS or BCG and then rested for 17 weeks before administrating the Th GP-FL 360 \nvaccine or PBS sham control.  Two weeks later, IFN ELISPOT assays were performed on 361 \nsplenocytes to determine recall responses of BCG specific Th cell against the peptide-25 (P25) of the 362 \nimmunodominant Ag85B or Mtb (strain H37Rv) lysate (Figs. 2A and B).  Mice in both of the BCG 363 \nvaccinated groups developed BCG specific Th cells reactive to Mtb lysate, but only the BCG group 364 \nthat was subsequently immunized with the Th GP-FL vaccine showed significant expansion of P25 365 \nspecific Th cells. 366 \nTo test the immunogenicity of the full length Th GP-FL vaccine and compare this directly 367 \nwith the MLD version (Th GPM) that we previously showed to lower the vaccine dose required to 368 \ninduce antibody responses and induce IgG2c class switching 33, mice were vaccinated with BCG or 369 \nreceived sham vaccination with PBS injection, and then primed and boosted by subcutaneous 370 \ninjections of either the Th GP-FL or Th GPM in alum.  A solid phase ELISA was performed to 371 \ndetect the presence of anti-EBOV GP-IgG1 and -IgG2c antibodies. Confirming our previously 372 \npublished findings 33, the BCG-specific Th cells from prior BCG vaccination, which were absent in 373 \nthe sham vaccinated (PBS) groups, were recruited by the Th vaccine to promote class switching to 374 \nIgG2c, and either version (Th GPM or Th GP-FL) of the Th vaccine induced similar antibody levels 375 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n9 \n(Fig. 2C).  Taken together, these results showed that the Th GP-FL can be used to replace the Th 376 \nGPM version of the vaccine to more accurately represent the native form of GP associated with 377 \nactual EBOV infection and provide the broadest array of potential epitopes for both neutralizing and 378 \nnon-neutralizing antibodies. 379 \n 380 \n 381 \nBCG vaccination upregulates FcRIV expression and supports long-lived antibody responses. 382 \n 383 \nNon-neutralizing antibodies mediate their functions primarily through the binding of FcRs to recruit 384 \nimmune cell effector functions, including cytolysis and phagocytosis, to clear infected cells.  In mice, 385 \nthe Fc portions of the IgG2 isotypes have the highest affinities for FcRIV, which is abundantly 386 \nexpressed on monocytes, macrophages, and neutrophils 31, and to a lesser degree on NK cells 56,57.  387 \nTo determine the expression level of FcRIV on immune cells, flow cytometry was performed to 388 \nidentify B cells (B220+) NK cells (NK1.1+), neutrophils (CD11bhigh Ly6Ghigh), macrophages 389 \n(CD11bhigh Ly6Chigh) and monocytes (CD11bhigh Ly6Clow) (Fig. 3A).  At 2 weeks after BCG 390 \nvaccination, an increase in the levels of FcRIV was detected on monocytes, macrophages, NK cells, 391 \nand B cells (Fig. 3B).  Although the Th vaccine expanded the BCG memory Th cells (Fig. 2), this 392 \nwas not associated with further enhancement of the BCG induced FcRIV expression at 17 weeks 393 \nafter the initial BCG vaccination (Fig. 4A & B).  However, these findings showed that BCG 394 \nvaccination induced prolonged elevation of FcRIV expression on effectors cells, which is likely to 395 \nbe relevant to the efficacy of the Th vaccine design that favors the induction of IgG2c class-switched 396 \nantibodies 33. 397 \nTo determine the duration of the persistence of antibodies against EBOV GP in mice 398 \nreceiving the Th GP-FL vaccine, mice were either vaccinated with BCG or sham vaccinated (PBS 399 \nonly), and then immunized with 5 μg of Th GP-FL (Fig. 5).  In this experiment, a higher dose of the 400 \nTh GP-FL was given to the animal instead of the usual dose of 0.5 μg per mouse in order to elicit a 401 \ndetectable IgG2c response in the PBS group for comparison with the BCG group.  Serum samples 402 \nwere collected at times ranging from 2 to 39 weeks after the administration of the Th GP-FL vaccine 403 \nand analyzed by ELISA for anti-EBOV GP titers for both IgG1 and IgG2c subclasses.  Compared to 404 \nthe PBS group that lacked BCG specific Th1 cells, the intrastructural help provided by BCG specific 405 \nTh1 cells in the BCG vaccinated group promoted higher anti-EBOV GP titers.  Anti-EBOV GP 406 \nantibodies were detected even at week 39 after vaccination (Fig. 5A), and, at the same time, plasma 407 \ncells secreting these anti-EBOV GP antibodies were detected in bone marrow and not the spleen (Fig. 408 \n5B), indicating that long lived plasma cells induced by the Th vaccine can elicit long lasting 409 \nprotection. 410 \n 411 \n 412 \nBCG vaccination induced extrafollicular Th1 responses and altered germinal center formation. 413 \n 414 \nIn our previous publication, we showed that antibodies induced by the Th vaccine have different 415 \naffinities in the Fab region that correlated with IgG1 and IgG2c isotypes 33.  B cells that enter the 416 \ngerminal center (GC) form cognate interaction with T follicular helper (Tfh) cells, which are defined 417 \nby expression of CXCR5 and the lineage-defining transcription factor Bcl-6 58, and go through 418 \nmultiple rounds of affinity maturation to develop high affinity antibodies.    B cells that encounter 419 \nantigens outside of GCs undergo cognate interactions with non-Tfh cells such as Th1 cells, which 420 \nreduces affinity maturation but provides rapid protection in early stages of infection 59.  To visualize 421 \nthe location of BCG-specific Th cells within a secondary lymphoid organ after BCG vaccination, we 422 \nused adoptive transfer of GFP labelled CD4+ T cells expressing a TCR transgene specific for the P25 423 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 10 \nepitope of BCG Ag85B as previously described 33.  To compare the extent to which these adoptively 424 \ntransferred T cells remained extrafollicular or were capable of entering germinal centers, we 425 \ncompared mice vaccinated with BCG versus mice immunized with P25 peptide combined with 426 \nvarious adjuvants, including alum and a multicomponent formulation known as LASTS-C (lipid A, 427 \nSpan8, Tween 80 and CpG oligodeoxynucleotides) 43.  16 hours after immunization, splenocytes 428 \nwere isolated and analyzed by FACS with gating on CD4+ GFP+ cells (Fig. 6A; top panel).  Th1 429 \npolarization of the transferred P25-specific GFP+ CD4+ T cells as determined by Tbet expression was 430 \nstrongest in BCG vaccination as compared with other adjuvants (Fig. 6A; middle panel), whereas the 431 \nTfh polarization as shown by CXCR5 and Bcl-6 double staining was extremely low except in 432 \nanimals receiving vaccination with the LASTS-C adjuvant (Fig. 6A; lower panel), which correlates 433 \nwith its ability to induce strong neutralizing antibody responses 44. 434 \nTo further evaluate the effects of BCG vaccination on the functional outcomes of CD4+ T cell 435 \nresponses, we analyzed the localization of P25 specific T cells in the spleen by 436 \nimmunohistochemistry.  Naïve P25-specific GFP+ CD4+ T cells were transferred intravenously into 437 \nmice that were vaccinated 16 hours later with either BCG or the P25 peptide adjuvanted in LASTS-438 \nC.  Six days after vaccination, spleens were isolated, sectioned, and analyzed by 439 \nimmunohistochemistry with anti-GFP staining followed by H&E counter staining (Fig. 6B).  BCG 440 \nvaccination, as expected for strong Th1 biasing stimuli, diminished the formation of GCs (Fig. 6B; 441 \nleft panel) as compared to non-Th1 adjuvant such as LASTS-C (Fig. 6B; right panel), which was 442 \nquantified by counting the number of GC per follicle (Fig. 6B).  In BCG vaccinated mice, the GFP+ 443 \nCD4+ T cells, observed as brown precipitate of the 3,3-diaminobenzidine in the 444 \nimmunohistochemistry staining with anti-GFP conjugated with horse radish peroxidase, were present 445 \nin the white pulp areas (Fig. 6B; left panel) but nearly absent in the relatively scarce GCs (Fig. 6C; 446 \nleft enlarged panel).  In contrast, in the LASTS-C vaccination group (Fig. 6B; right panel) there were 447 \nnumerous GCs, and the GFP+ CD4+ T cells were mostly localized within the GCs (Fig. 6C; right 448 \nenlarged panel), as quantified as the number of P25 cells per GC (Fig. 6C).  These data suggested 449 \nthat the majority of BCG Th cells remained outside the GC at the border of the B cell follicle and 450 \nlikely exerted their effects on the antibody response at this location comprising the boundary of B 451 \nand T cell zones. 452 \n 453 \n 454 \nVaccination with subunit vaccine for protection against EBOV challenge. 455 \n 456 \nExperiments to assess our proposed regimen for protection from lethal EBOV infection required the 457 \nuse of mice that will have reached 24 weeks of age at the time of infection with EBOV, an age group 458 \nthat has not to our knowledge been previously tested in the EBOV challenge model.  To determine 459 \nwhether mice at this age have similar susceptibility to EBOV infection compared to the typically 460 \nused younger mice, 24-week-old mice were challenged with 10 or 1,000 focus-forming units (FFU) 461 \nof mouse-adapted EBOV (MA-EBOV).  Eight days after challenge, 24-week-old mice succumbed to 462 \nthe MA-EBOV infection even with the lower 10 FFU infectious dose (Supplemental Fig. 3), which 463 \nwas similar to the time to death observed previously in younger mice at 6-14 weeks of age 46.  464 \nHaving established the susceptibility of the older mice in this model, we tested the efficacy of our 465 \nregimen using recombinant protein vaccines augmented by prior BCG vaccination to protect against 466 \nEBOV challenge using the vaccination strategy illustrated in Figure 7A.  Sera were analyzed by 467 \nELISA for anti-EBOV GP antibody responses 2 weeks after priming and again after boosting with 468 \nthe subunit vaccines.  The group receiving the WT GP-FL vaccine (WT), which is incapable of 469 \nrecruiting BCG Th cells for intrastructural help, failed to induce robust anti-EBOV GP antibody 470 \nresponses, and did not undergo IgG2c class-switching (Fig. 7B, left).  This was in contrast to the Th 471 \nGP-FL vaccine (Th), which enhanced anti-EBOV GP antibody responses and IgG2c class-switching 472 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n11 \n(Fig. 7B, right).  An ELISA performed with end-point dilution of the serum samples collected after 473 \nthe Th-vaccine boost showed similar results, with the Th GP-FL vaccine providing elevated levels of 474 \nboth IgG1 and IgG2c antibodies against EBOV GP (Fig. 7C).  Five weeks after boosting with the 475 \nsubunit vaccines, mice were challenged with 10 FFU of MA-EBOV.  All the mice from the PBS and 476 \nthe WT GP-FL vaccine group succumbed to the infection 8 days after MA-EBOV challenge, whereas 477 \nthe majority of the mice in the Th GP-FL vaccine group survived through the end of the study, at 478 \nwhich point they appeared healthy and had regained their original body weight (Fig. 7D).  In 479 \naddition, at day 5 after EBOV infection, five mice were sacrificed to determine the viral titers in the 480 \nblood and organs.  Mice that received the Th GP-FL vaccine had a lower viral titer in the blood, liver, 481 \nand spleen when compared to mice that either received no vaccination or the WT GP-FL (Fig. 7E).  482 \nAn ELISA was also performed on the serum samples collected from these mice which showed that 483 \nmice that received the Th GP-FL vaccine also had a higher anti-EBOV GP antibody titer compared to 484 \nthe control group receiving PBS only or the WT GP-FL vaccine group (Fig. 7F).  Long term 485 \nsurvivors from the Th vaccine group were also bleed at termination of the experiment (day 112, 486 \ncorresponding to 14 days after EBOV challenge), and analysis of these serum samples showed 487 \npersistently high levels of anti-EBOV GP antibodies (Fig. 7G).  Thus, the Th vaccine strategy clearly 488 \nprotected the mice against lethal EBOV infection by limiting viral replication to control the early 489 \nstage of infection, which is known to be important in conferring protection as seen in other viral 490 \ninfections 59.  491 \n 492 \n 493 \nThe approach to antiviral vaccination used in the current study is based on the classic hapten-carrier 494 \nimmunization studies that led to the understanding of the concept of linked recognition.  Many 495 \neffective vaccines depend on the core immunological concept of linked-recognition, in which Th 496 \ncells recognize processed peptides derived from the immunogen targeting B cell receptors to provide 497 \nintrastructural help to B cells, leading to T cell dependent antibody responses 60.  These include 498 \nvaccines that rely on the production of antibodies against targets that entirely lack T cell epitopes, 499 \nsuch as those against Haemophilus influenzae type b (Hib) polysaccharides 61 or small hapten-like 500 \nmolecules like nicotine 62.  In these cases, conjugation to a protein carrier containing Th cell epitopes 501 \nis required to elicit an optimal T cell dependent B cell response.  In a logical extension of this 502 \nprinciple, we and others have applied this approach to creating protein subunit vaccines against viral 503 \nantigens to enhance and accelerate protective antibody responses through the recruitment of pre-504 \nexisting Th cells against other potent antigens, such as those delivered by previous vaccination 505 \nagainst pathogens such as mycobacteria.  For example, Klessing et al. developed a vaccine against 506 \nHIV that can recruit intrastructural help from Th cells induced by an M. tuberculosis subunit vaccine, 507 \nand showed that this approach induced higher antibody titers that persisted for extended period of 508 \ntime 63.  In our previous work we applied a similar approach to capture intrastructural help to B cells 509 \nfrom pre-existing Th1 cells specific for immunodominant mycobacterial antigens in BCG vaccinated 510 \nmice 33.  In the current study, we expanded on our previous work to determine the protective efficacy 511 \nof this Th vaccine design against EBOV challenge in the mouse model, and to explore in greater 512 \ndetail the potential mechanisms mediating this protection.  Consistent with our findings, we showed 513 \nthat this vaccine strategy induced antibody class-switching to IgG2c, an isotype that is known to have 514 \nhigh affinity toward FcRIV, suggesting that antibodies with effector activities such as antibody-515 \nmediated cellular cytotoxicity (ADCC) might be a key feature that extended the antiviral effects 516 \nbeyond simple neutralization of viral entry.  517 \nOur previous efforts to generate fusion proteins for use as subunit vaccines against EBOV 518 \nused a truncated form of EBOV GP in which the MLD was deleted, which our preliminary work had 519 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 12 \nshown to be produced with much higher yields than a full-length version (Th GP-FL) that retains the 520 \nMLD  33.  In the current study, we further improved production and purification of the Th GP-FL 521 \nfusion protein and formulated this with alum to generate a candidate vaccine against EBOV infection 522 \nand disease for use in previously BCG vaccinated hosts. This full-length version of the extracellular 523 \ndomains of EBOV GP should present an immunogen that corresponds more closely than the 524 \npreviously designed Th GPM vaccine that lacks the MLD to the actual infectious virus or the form 525 \nof the EBOV GP expressed on the surface of infected host cells, making it potentially more effective 526 \nfor generating a broad range of antibodies mediating a variety of host protective functions.  In this 527 \nregard, while epitopes in the MLD region have not been strongly associated with broad neutralization 528 \nof viral entry, such antibodies may play an important role in controlling the progression and spread of 529 \ninfection through non-neutralizing activities such as ADCC 64. 530 \nOur preparations of the Th GP-FL fusion protein produced so far appeared to exist mainly as 531 \nhigher order multimers in solution, rather than as soluble monomers or native homotrimers 532 \n(Supplemental Fig. 1).  The multimerization of the protein may be due to artifactual disulfide bond 533 \nformation or other tight interactions that formed during the purification, and suggests the need for 534 \nfurther optimization of the production and purification process.  However, irrespective of the 535 \npresence of larger multimeric complexes in the Th GP-FL preparations, the native conformation of 536 \nthe protein appeared to be present at significant levels, as shown by its recognition by the anti-EBOV 537 \nGP monoclonal antibodies, ADI-15878 and KZ52, which recognize conformational epitopes of the 538 \nnative protein (Fig. 1C and Supplemental Fig. 2). Furthermore, the Th GP-FL vaccine was able to 539 \ninduce anti-EBOV GP antibodies that recognized EBOV GP expressed on the surface of recombinant 540 \nvesicular stomatitis virus (Figs. 2C, 5A, and 7B).  Most importantly, the vaccine conferred protection 541 \nagainst challenge with MA-EBOV (Fig. 7D & E), indicating that antibodies generated by the Th GP-542 \nFL vaccine, particularly when administered in the context of prior BCG vaccination, were able to 543 \nrecognize the relevant form of GP during viral infection. 544 \nA key feature of our vaccine strategy is the prior BCG vaccination, which not only induced 545 \nmemory BCG-specific Th1 cells to provide intrastructural help, but also through trained immunity, 546 \ncan enhance non-specific immune mechanisms 65.  Protection from trained immunity induced by 547 \nBCG has been described in COVID-19 infection and also in BCG-based bladder cancer treatments 66.  548 \nIn our analyses, we observed that BCG exposure also promoted FcRIV expression (Figs. 3 and 4) 549 \nand IgG2c class-switching (Fig. 2C), which can be viewed as additional aspects of trained immunity.   550 \nIn the mouse model, the IgG2c isotype and FcRIV expression together are important for the 551 \ninduction of ADCC by certain immune effector cells such as neutrophils and NK cells.  Correlating 552 \nwith the induction of anti-EBOV GP IgG2c antibodies together with FcRIV expression, BCG-553 \nvaccinated mice that received the Th GP-FL vaccine, but not those with the WT GP-FL vaccine, 554 \nsurvived the EBOV challenge (Fig. 7D). This suggests that the anti-EBOV GP IgG2c isotype (Fig. 555 \n7B) played a significant role in conferring this protection.  Analyzing the location of the BCG Th1 556 \ncells revealed that the anti-EBOV GP IgG2c antibodies were likely derived from extrafollicular 557 \nplasmablasts since BCG vaccination induces a strong Th1 cell response that favor less GC 558 \ndevelopment as compared to a Tfh promoting adjuvant (Fig. 6).  As a result of this massive Th1 559 \npolarization, most of the T-dependent B cells are activated by the Th1 cells at the boundary of B cell 560 \nfollicles and not inside GCs.  These GC-nonresident B cells develop into extrafollicular plasmablasts 561 \nwhich are usually short-lived.  Surprisingly, at 39-weeks after vaccination anti-EBOV GP antibody 562 \nlevels were still detected in vaccinated mice (Fig. 5A) and the EBOV GP-specific antibody secreting 563 \ncells were still detected in the bone marrow (Fig. 5B), presumably representing long-lived plasma 564 \ncells.  This suggests the possibility that GC in BCG vaccinated mice, although not initially detected, 565 \nmay form at a later time and enable extrafollicular B cells induced in early stages post-vaccination to 566 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n13 \ndevelop into memory B cells and long-lived plasma cells that take up residence in the bone marrow 567 \nto sustain long term production of anti-EBOV GP antibodies. 568 \nAlthough other forms of EBOV vaccine are available such as the VSV-based EBOV vaccine 569 \n(Ervebro) that is FDA approved for human use 67-69 and confers protection in non-human primates 10 570 \ndays after vaccination 70, the disadvantages faced by virus vectored EBOV vaccines are 571 \nmanufacturing difficulties for large scale production and cold chain requirement during distribution 572 \n69, which can easily overwhelm logistics when dealing with larger outbreaks.  Other EBOV vaccine 573 \nplatforms require strong adjuvants to increase immunogenicity 49,50,71, including recombinant subunit 574 \nvaccines based on EBOV GP that are currently being developed 49,50,71.  The development of a 575 \nsubunit EBOV vaccine should allow easier production and distribution, especially in resource limited 576 \nnations, which can contribute to rapid deployment to control outbreaks 72.  The recombinant protein 577 \nvaccine used in this study has the unique ability to recruit BCG-specific Th1 cells to provide 578 \nintrastructural help for driving antibody production against the recombinant protein subunit without 579 \nthe use a strong adjuvant that can increase cost and unwanted side effects.  These properties can also 580 \nlower the dose of the recombinant vaccine required, which can have an impact on manufacturing, 581 \ncost, and distribution worldwide.  Furthermore, sustained antibody responses through week 39 was 582 \nobserved after administering a single dose of the Th GP-FL vaccine.  The benefit of the Th GP-FL 583 \nvaccine developed in this study as a recombinant protein, no doubt, is its simplicity as compared to a 584 \nvirus vaccine, and its ability to harness pre-existing BCG-induced immunity to protect mouse against 585 \nMA-EBOV infection.  Based on current projections 73, BCG vaccination will continue in many 586 \nregions of the world well into the future 74, thus establishing large populations that should be well 587 \nsuited for mass vaccination against EBOV or other emerging viruses 75 using the approach 588 \ndemonstrated by the current study. 589 \n 590 \n5 Conflict of Interest 591 \nThe authors declare that the research was conducted in the absence of any commercial or financial 592 \nrelationships that could be construed as a potential conflict of interest. 593 \n 594 \n6 Author Contributions 595 \nTWN and SAP: experimental conception, design, analysis, interpretation of data, and writing of the 596 \nmanuscript. TWN: performed experiments and the analysis and acquisition of data.  WF and AM: 597 \ndesign, performed, acquired, and analyzed data for the EBOV mouse challenge.  ASW: prepared the 598 \nrVSV EBOV GP.  NASA: assisted with analysis of histology data of spleen sections.  CTJ: assisted 599 \nwith the FACS analyses.  AM, WRJ, and KC: analyzed, interpreted experiments, and reviewed the 600 \nmanuscript. All authors critically reviewed and approved the manuscript. 601 \n 602 \n7 Funding 603 \nThe core facilities used in this study were all supported in part by NCI Cancer Center Service Grant 604 \nP30CA013330.  Shared instrumentation grants funded the purchase of the Cytek Aurora FACS 605 \nanalyzer (S10OD026833-01) and the 3DHistec Panoramic 250 Flash II slide scanner 606 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 14 \n(1S10OD019961-01) used in this study.  This study was in part supported by the Intramural Research 607 \nProgram, NIAID, NIH (AM). 608 \n 609 \n8 Acknowledgments 610 \nResources and advice were provided by core facilities at Albert Einstein College of Medicine, 611 \nincluding the Flow Cytometry, Analytic Imaging and Histopathology facilities.  We thank Dr, Scott 612 \nGarforth and the Macromolecular Therapeutics Development Facility at Albert Einstein College of 613 \nMedicine for performing the size exclusion chromatography.  We also thank Mei Chen and John Kim 614 \n(Department of Microbiology & Immunology, Albert Einstein College of Medicine) for expert 615 \ntechnical assistance with mouse experiments.  We thank Bing Chen (Department of Microbiology & 616 \nImmunology, Albert Einstein College of Medicine) for assistance in maintaining of FcR KO mice 617 \ncolony.  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Vaccines (Basel) 9 808 \n(2021). https://doi.org:10.3390/vaccines9030190 809 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n19 \n71 Agnolon, V. et al. Designs and Characterization of Subunit Ebola GP Vaccine Candidates: 810 \nImplications for Immunogenicity. Front Immunol 11, 586595 (2020). 811 \nhttps://doi.org:10.3389/fimmu.2020.586595 812 \n72 Ng, T. W. & Porcelli, S. A. Designing Anti-Viral Vaccines that Harness Intrastructural Help 813 \nfrom Prior BCG Vaccination. J Cell Immunol 5, 97-102 (2023). 814 \nhttps://doi.org:10.33696/immunology.5.174 815 \n73 Scriba, T. J., Netea, M. G. & Ginsberg, A. M. Key recent advances in TB vaccine 816 \ndevelopment and understanding of protective immune responses against Mycobacterium 817 \ntuberculosis. Semin Immunol 50, 101431 (2020). https://doi.org:10.1016/j.smim.2020.101431 818 \n74 Cernuschi, T., Malvolti, S., Nickels, E. & Friede, M. Bacillus Calmette-Guerin (BCG) 819 \nvaccine: A global assessment of demand and supply balance. Vaccine 36, 498-506 (2018). 820 \nhttps://doi.org:10.1016/j.vaccine.2017.12.010 821 \n75 Gayer, M., Legros, D., Formenty, P. & Connolly, M. A. Conflict and emerging infectious 822 \ndiseases. Emerg Infect Dis 13, 1625-1631 (2007). https://doi.org:10.3201/eid1311.061093 823 \n 824 \n10 Figure Captions 825 \n 826 \nFigure 1.  Characterization and comparison of EBOV GP vaccines.   (A) Schematic of the Th 827 \nGP-FL vaccine against EBOV.  The Th vaccine consist of the N-terminal human Ig-kappa signal 828 \nsequence (hIgKss), the BCG T helper epitopes (P25 and P10) which are flanked by the cathepsin B 829 \ncleavage site (TVGL), GP1 which includes the mucin-like domain (MLD), the furin cleavage site, 830 \nGP2, and the C-terminal hexahistadine tag (6xHis).  GP1 and GP2 are held together by a disulfide 831 \nbond.  For the WT GP-FL version of the vaccine, the BCG T helper epitopes (P25 and P10) flanked 832 \nby the cathepsin B cleavage sites are absent.  The MLD deleted versions of these vaccines were also 833 \nconstructed (Th GPM and WT GPM).  (B) SDS-PAGE under reducing conditions of purified 834 \nEBOV GPs as shown by Coomassie gel staining and Western blotting with anti-His antibody.  The 835 \nGP1 and GP2 fragments, which are normally held together by disulfide bonds, were separately 836 \nresolved under the reducing and denaturing conditions of the SDS-PAGE analysis.  The expected 837 \nsize for GP2, which consists of the C terminal region of the EBOV GP after the MLD is 25 KDa.  838 \nThe expected size for the GP1 precursor is 120 KDa and 60 KDa for the full length and the MLD 839 \ndeleted version of the EBOV GP, respectively.  The GP0 fragment in the full-length versions of the 840 \nGP constructs was detected as two or more bands of ~120-145 KDa, consistent with glycosylation 841 \nand disordered structure of the MLD.  Purity of isolated GPs was ≥ 90% based on Coomassie blue 842 \nstaining of the gels, and protein yields were determined by BCA protein assay (WT GP-ΔM: 2060 843 \nμg/ml, WT GP-FL: 220 μg/ml, Th GP-ΔM: 2972 μg/ml, Th GP-FL: 80 μg/ml).  (C) ELISA with 844 \nantibody ADI-15878 specific for EBOV GP conformational epitope was used to probe purified 845 \nEBOV GPs.  The ovalbumin version of the Th vaccine (Th OVA) served as a negative control to 846 \nshow the specificity of ADI-15878 antibody against EBOV GP.  (D) Processing and presentation of 847 \nBCG Th epitopes was shown by incubating purified EBOV GPs for 16 hours with dendritic cells and 848 \nin the presence of a CD4+ T cell hybridomas specific for P25 of Ag85B (left) or P10 of TB9.8 (right).  849 \nSupernatants were analyzed by sandwich ELISA for IL-2 (indicated as absorbance (Abs) values for 850 \nconversion of the assay substrate. Multiple columns were analyzed by Kruskal-Wallis one-way 851 \nANOVA, followed by Dunn’s multiple comparison test; (***p < 0.001, **p < 0.01, *p < 0.05).   852 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 20 \n 853 \nFigure 2.  Induction of cellular and humoral immune responses by Th vaccines.  For analysis of 854 \ncellular responses, groups of mice (n = 5) were vaccinated with BCG or received sham vaccination 855 \nwith PBS injection, rested for 17 weeks and then immunized with the Th GP-FL or sham immunized 856 \n(PBS).  Two weeks after the immunization, IFN ELISPOT assays were performed on unstimulated, 857 \npeptide-25 (P25) or Mtb (H37Rv lysate) stimulated splenocytes.  (A) Representative spot forming 858 \ncell (SFC) images of selected animals for each group.  (B) Plots showing individual animal counts 859 \nand group medians with interquartile range.  Multiple columns were analyzed by Kruskal-Wallis one-860 \nway ANOVA, followed by Dunn’s multiple comparison test; (***p < 0.001 and **p < 0.01).  Note 861 \nthat values for H37Rv lysate stimulation of BCG vaccinated groups are all plotted at the upper limit 862 \nfor accurate quantitation in the assay.  (C) Mice (n = 5) were vaccinated with BCG or received sham 863 \nvaccination with PBS injection, rested for 5 weeks and then prime and boosted with the EBOV GP 864 \nvaccines (Th GP-FL or Th GPM).  Two weeks after the boost, sera were collected, and antibody 865 \ntiters against EBOV GP (WT FL) were determined using ELISA specific for IgG1 or IgG2c isotypes. 866 \n 867 \nFigure 3.  Expression of FcRIV increases at week 2 after BCG vaccination. (A) Flow cytometry 868 \ngating strategy using mice with compound genetic knock out of FcRII, RIII and RIV -chains (KO) 869 \nand wildtype (WT) C57BL/6 mice to determine the gating for FcRII/III and FcRIV expression on 870 \nimmune cells.  After singlet cell gating, the corresponding surface markers were used to stain 871 \nsplenocytes to identify the following immune cells: monocytes (CD11b+ Ly6Clow), macrophages 872 \n(CD11b+ Ly6Chigh), neutrophils (CD11b+ Ly6G+), NK cells (NK1.1+), and B cells (B220+). (B) Mice 873 \n(C57BL/6) were vaccinated with 107 BCG per mouse or received PBS injections as control.  Spleens 874 \nwere harvested at week 1 after BCG vaccination (gray histogram), or at week 2 after PBS injection 875 \n(white histogram) or BCG (black histogram) vaccination, and splenocytes were analyzed by FACS to 876 \ndetermine the expression level of FcRIV. Top panel shows representative histograms for an 877 \nindividual mouse from each group, and bottom panel shows median of MFI values for 5 mice in each 878 \ngroup on each indicated cell type.  Median with interquartile range for five replicates is shown and 879 \nresults were analyzed using Kruskal-Wallis one way ANOVA nonparametric test and Dunn’s 880 \nmultiple comparison test; (***p < 0.001, **p < 0.01). 881 \n 882 \nFigure 4.  BCG-induced FcRIV expression was maintained after Th vaccination.  Mice (n = 10) 883 \nwere sham vaccinated with PBS or with BCG at 107 CFU per mouse and rested for 17 weeks.  Each 884 \ngroup was further subdivided into 2 groups (n =5) and received either PBS or the Th GP-FL vaccine 885 \nat 0.05 μg per mouse in alum, and splenocytes were harvested 2 weeks later to determine the level of 886 \nFcRIV expression by FACS.  Singlet gating on splenocytes were stained for macrophages 887 \n(CD11bhigh Ly6Chigh), neutrophils (CD11bhigh Ly6Ghigh), and NK cells (NK1.1high) for expression of 888 \nFcRIV (CD16.2) as shown in (A) for representative animals of sham vaccinated with PBS and with 889 \nBCG alone, and quantified in (B) by MFI levels for the 5 animals in each group.  The median with 890 \ninterquartile is shown and analyzed using Kruskal-Wallis one way ANOVA nonparametric test with 891 \nand Dunn’s multiple comparison test; (**p < 0.01 and *p < 0.05). 892 \n 893 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n \n21 \nFigure 5. Long lasting humoral immune response induced by the Th vaccine.  (A) Serum 894 \nsamples from sham PBS () or BCG (•) vaccinated mice and subsequently immunized once with 5 895 \nμg of the Th GP-FL vaccine were analyzed by ELISA for anti-EBOV GP IgG1 (left panel) or IgG2c 896 \n(right panel) antibodies throughout the time course of 39 weeks.  Serum from a naïve mouse () 897 \ncollected at week 2 and 4 was used to measure the background level for the ELISA assay.  (B) 898 \nSplenocytes and bone marrow cell suspensions from these mice at 39 weeks were harvested and used 899 \nfor B cell ELISPOT assay to quantitate anti-EBOV GP IgG1 or IgG2c antibody secreting spot 900 \nforming cells (SFCs).  The median values with interquartile ranges are shown and analyzed using 901 \nKruskal-Wallis one way ANOVA nonparametric test with Dunn’s multiple comparison test; (*p < 902 \n0.05).  903 \n 904 \nFigure 6. BCG vaccination promotes predominantly Th1 responses.  (A) Wildtype mice were 905 \nadoptively transferred with 4 x 104 T cells purified from P25 TCR-Tg GFP+ mice 16 hours prior to 906 \nvaccination with BCG (B), or peptide-25 adjuvanted with either alum (A) or LASTS-C (LC).  The 907 \nmice were sacrificed 6 days after vaccination, and splenocytes (n=5) were analyzed by FACS for 908 \nmaster transcription regulators and key markers for Th1 (Tbet) and Tfh (CXCR-5 and Bcl-6).  909 \nMultiple comparisons were analyzed by Kruskal-Wallis one-way ANOVA (**p < 0.01, ***p < 910 \n0.001). (B) Wildtype mice were adoptively transferred with 4 x 104 T cells purified from P25 TCR-911 \nTg GFP+ mice 16 hours prior to vaccination with BCG or with the P25 peptide adjuvanted with 912 \nLASTS-C.  Formalin-fixed and paraffin-embedded spleens were cut into thin sections for 913 \nimmunocytochemistry with anti-GFP followed by hematoxylin and eosin counterstaining.  (C) High 914 \nmagnification of boxed areas in (B) to visualize the P25 specific T cells as dark colored spots.  915 \nFollicles (F), germinal centers (GC), and P25 Th cells were quantified manually by a blinded 916 \nobserver as the number of GC per follicle in (B), and the number of P25 Th cells per GC in (C).  917 \nMedians with interquartile ranges for 6-7 sections derived from 3 mice from each group are plotted.  918 \nMann-Whitney test was used for pairwise comparison (**p < 0.01, ***p < 0.001). 919 \n 920 \nFigure 7. Vaccine induced protection against lethal EBOV infection.  (A) Timeline of the EBOV 921 \nchallenge experiment.  C57BL/6NHsd mice (n = 15) were vaccinated with BCG, and subsequently 922 \nprimed and boosted with 0.05 μg of the indicated recombinant protein vaccine (WT GP-FL or the Th 923 \nGP-FL) in alum as indicated in the schematic.  MA-EBOV infection (i.p.) with 100 pfu per mouse 924 \nwas performed on day 98, which corresponds to day 0 (blue) of the challenge experiment.  Arrows 925 \nrepresent blood sample collections, and harvesting of blood, livers, and spleens at the last collection 926 \npoint after sacrifice.  (B) Serum samples collected 2 weeks after administering WT GP-FL (WT) or 927 \nthe Th GP-FL (Th) vaccines for both priming and boosting time-points were analyzed by ELISA for 928 \nanti-EBOV GP IgG1 (red) and IgG2c (blue) antibodies.  (C) Endpoint titers of anti-EBOV GP 929 \nantibodies following boosting with the WT or Th vaccines were also analyzed by ELISA in sera from 930 \nmice primed and boosted with either PBS or 0.05 μg of the indicated recombinant protein vaccine.  931 \n(D) Naïve mice (black) or mice that received the WT (red) or Th (blue) vaccines were challenged 932 \nwith MA-EBOV and the times to death (left panel) and body weight changes (right panel) for 933 \nindividual mice were recorded.  The Mantel-Cox test was performed for comparison of the survival 934 \ncurves; (****p < 0.0001).  (E) Five days after the challenge, mice (n = 5) were sacrificed to 935 \ndetermine viral titers from the corresponding organs.  The median with interquartile ranges is shown 936 \nand analyzed using Kruskal-Wallis one way ANOVA nonparametric test with and Dunn’s multiple 937 \ncomparison test; (**p < 0.01, *p < 0.05).  (F) The anti-EBOV GP IgG1 and IgG2c antibody titers 938 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\n 22 \nwere also measured from serum of these mice sacrificed at day 5 after challenge (****p < 0.0001, 939 \n**p < 0.01, *p < 0.05 two-way ANOVA test and Sidak’s multiple comparison test).  (G) Twenty-940 \neight days after the challenge, the surviving mice were sacrificed, and blood was collected to measure 941 \nanti-EBOV GP IgG1 and IgG2c antibody titers.   942 \n 943 \n11 Supplementary Material Captions 944 \n 945 \nSupplemental Figure 1.  Analysis of EBOV GP by size exclusion chromatography.  Purified 946 \nwildtype EBOV GP full-length (WT GP-FL) and the Th EBOV GP vaccine (Th GP-FL) were 947 \nsubjected to the SRT SEC-300 size exclusion column.  SRT SEC-300 exclusion limit is 1250 kDa. 948 \n 949 \nSupplementary Figure 2. Comparison of EBOV GP vaccines.  (A) ELISA with antibody KZ52 950 \nspecific for EBOV GP conformational epitopes was used to probe purified EBOV GP.  The 951 \novalbumin version of the Th vaccine (Th OVA) served as a negative control to show the specificity 952 \nof KZ52 antibody against EBOV GP (left panel). 953 \n 954 \nSupplemental Figure 3.  Susceptibility of 24-week old C57BL/6N to MA-EBOV challenge.  24-955 \nweek old mice were infected with 10 or 1000 FFU of mouse-adapted (MA-) EBOV and succumbed 956 \nto infection by day 7-8 after EBOV challenge.  957 \n 958 \nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint \n\nwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}