A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus

Ebola virus, Mycobacterium bovis BCG, CD4+ T cells, antibodies, vaccines, 14 intrastructural help, Fc receptors. 15

16 Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that 17 takes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille 18 Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion 19 proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, 20 engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by 21 prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, 22 we show that Th cells induced by BCG were localized predominantly outside of germinal centers and 23 promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and 24 effector functions. Furthermore, BCG vaccination also upregulated FcR expression to potentially 25 maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) 26 infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and 27 protection against lethal challenge. This general approach can be easily adapted to other viruses, and 28 may be a rapid and effective method of immunization against emerging pandemics in populations 29 that routinely receive BCG vaccination. 30 1 Introduction 31 Emergent viruses such as Ebola virus (EBOV) and related filoviruses are global health threats of 32 increasing concern, especially due to the expansion of human populations into wild habitats that 33 serve as natural reservoirs for these viruses 1. For prevention of outbreaks of viral infections or 34 pandemics, vaccines remain the most practical and cost-effective tools. This has clearly been shown 35 in the ongoing Coronavirus disease 2019 (COVID-19) pandemic where vaccination has been 36 reported to reduce the risk of severe illness leading to hospitalization and mortality rates among 37 vaccinated individuals 2. Historically, vaccine development has been mainly focused on the variable 38 (Fab) region of immunoglobulins for their ability to bind surface antigens of viruses and prevent 39 entry into host cells 3-5. Such neutralizing antibodies (NAbs) are a major correlate of protection 40 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 2 associated with viral clearance and resolution of the infection. However, the limited range of 41 epitopes available to induce NAbs may prevent efficient clearance of infection by this mechanism for 42 some viruses. Furthermore, as shown in human immunodeficiency virus (HIV) infection, NAbs exert 43 strong selective pressure in driving immune escape of the virus as compared to non-neutralizing 44 antibodies 6. These observations suggest that eliciting non-neutralizing antibodies mediating effector 45 functions distinct from simple blockade of viral entry might increase the protective efficacy of a 46 vaccine 7,8. 47 The constant (Fc) regions of antibodies, although recognized as important in contributing to 48 protection, have been less emphasized compared to the Fab region as a determinant of anti-viral 49 effects. The Fc region binds to the Fc receptors (FcRs) on a variety of relevant immune effector 50 cells, thus bridging humoral and cellular immunity through effector activities such as antibody-51 dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-52 dependent cellular cytotoxicity (ADCC) that are believed to contribute to control of viral and other 53 microbial infections 9,10. Recently, results from clinical trials for newly developed EBOV and HIV 54 vaccines have called attention to the importance of antibody-mediated effector functions as correlates 55 of protection against viral pathogens 11-15. This has driven the search for relevant antibody effector 56 functions beyond simple neutralization in individuals vaccinated against or exposed to EBOV 16,17 or 57 HIV 18,19, and has encouraged efforts to engineer therapeutic antibodies with optimal Fc effector 58 functions for these diseases 20,21. 59 Whereas the binding affinity of the Fab region of the antibody develops and matures in the 60 germinal centers (GC) within B cell follicles of secondary lymphoid tissues 22, class-switch 61 recombination (CSR) required for determining Fc isotype is initiated and occurs mostly at the border 62 of the B cell follicle between the boundary of B and T cell zones 23. Activation of CSR requires 63 signals from B cell receptor (BCR) engagement, costimulatory signals such as CD40-CD40L 64 interaction, and particularly cytokines secreted from CD4+ helper T cells (Th) that dictate which 65 switch region of the heavy chain constant region genes will interact with activation-induced cytidine 66 deaminase (AID) to initiate the double strand DNA break required for recombination to occur 24. 67 Therefore, the ability to induce different Th phenotypes, such as Th1, Th2 or follicular helper T cells 68 (Tfh), during vaccination can have an impact on class-switching of immunoglobulins. In C57BL/6 69 mice, for example, class-switching to IgG2c (homologous to IgG2a in other mouse strains 25), is 70 induced by IFN produced by Th1 cells 26,27, whereas IgG1 is induced by IL-4 derived mainly from 71 Th2 cells 28-30. These antibody subclasses have different affinities to particular Fc receptors (FcRs) 72 31. In mice, antibodies with the IgG1 isotype have low, but similar affinities for the inhibitory 73 FcRIIB and the activating FcRIII, whereas the affinities conferred by the IgG2 isotypes for the 74 activating FcRIV are much stronger 31. Under inflammatory Th1 conditions, IgG2c class switching 75 and an increased expression of FcRIV are favored 32, thus promoting effector functions such as 76 phagocytosis, complement activation and cytotoxicity, all contributing to the removal of either the 77 pathogen itself or the cells infected by it. 78 Mycobacterium bovis bacille Calmette-Guérin (BCG), the only currently approved vaccine 79 against tuberculosis, is one of the most widely administered vaccines in many regions of the world. 80 The BCG vaccine induces long lasting BCG-specific memory CD4+ T helper cells (Th) that are 81 strongly polarized to IFNγ-secreting Th1 cells in vaccinated individuals. To take advantage of the 82 high prevalence of BCG vaccination, we developed a vaccination strategy that uses pre-existing 83 BCG-specific Th cells to drive antibody responses against a modified viral protein immunogen 33. 84 This recombinant fusion protein vaccine (Th-vaccine), based on a principle previously designated 85 intrastructural help 34,35, induces antiviral antibody responses with a strong bias to IgG2c isotype in 86 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 3 C57BL/6 mice 33. In the current study, we have used an EBOV challenge model to show that this 87 approach promotes antibodies that can recruit effector cells capable of eliminating virus infected cells 88 and is highly effective at protecting mice from lethal virus challenge. Analysis of the underlying 89 mechanism for the effects on the antibody response showed that BCG vaccination created 90 inflammatory conditions that impaired GC formation, similar to what has been seen in infections 91 with other Th1 skewing pathogens 36-39. Antibody class switching thus occurred outside of the GC, 92 leading to a strong bias of anti-EBOV antibodies to IgG2c isotype due to the influence of BCG-93 specific Th1 cells. The anti-EBOV GP antibodies elicited by this vaccination regimen were 94 maintained over time suggesting the induction of long-term protection. Overall, our findings support 95 the importance of non-neutralizing antibodies in anti-viral vaccination, and define a powerful and 96 potentially useful method to induce such antibodies against established or newly emerging viruses in 97 populations that receive routine BCG vaccinations. 98 2 Materials and Methods 99 Mice 100 101 Five-week old female wild-type (WT) C57BL/6NHsd and C57BL/6J mice were obtained from 102 Envigo (Greenfield, IN) and The Jackson Laboratory (Bar Harbor, ME), respectively. The GFP+ 103 C57BL/6-P25 TCR transgenic (Tg) mice 33 with T cell receptor that recognizes the P25 peptide 104 (FQDAYNAGGHNAVF) from M. tuberculosis or BCG Ag85B were maintained and bred in our 105 facility. All mice were maintained in our specific pathogen-free facilities following protocols and 106 regulations established by the Albert Einstein College of Medicine Institutional Animal Use and Care 107 and the Institutional Biosafety Committees. All procedures performed on these animals were 108 approved by the Albert Einstein College of Medicine Institutional Animal Use and Care Committee. 109 110 111 Mycobacterial strains and vaccinations 112 113 Mycobacterium bovis BCG Danish strain (Statens Serum Institut, Copenhagen, Denmark) was the 114 BCG vaccine strain used in this study. Starting from a low-passage-number frozen stock, the 115 bacteria was grown at 37C shaking in Sauton medium until mid-log phase, centrifuged at 600 x g for 116 10 minutes, and resuspended in sterile PBS (Thermo Fisher Scientific, Waltham, MA). BCG was 117 administered by subcutaneous (s.c.) injection at the scruff of the neck at a dose of 1 x 107 CFU. For 118 recombinant protein vaccines injections, the vaccine in PBS was mixed in a 1:1 volume ratio with 119 alum suspension (Imject Alum; Thermo Fisher Scientific) to a final concentration of 0.5 µg/ml unless 120 otherwise specified, and 100 µl was administered intramuscular (i.m.) into the thigh muscles with 50 121 µl per hind limb to provide the final dose of 0.05 µg of the recombinant protein vaccine per animal. 122 123 124 Cell lines 125 126 FreeStyle 293-F cells (Thermo Fisher Scientific) were maintained in Life Technologies FreeStyle 127 293 Expression Medium with GlutaMAX (Thermo Fisher Scientific). Murine T cell hybridomas 128 (TCHs) specific for I-Ab -restricted CD4 T cell epitopes 33 were maintained in complete RPMI 129 (cRPMI) which consists of RPMI 1640 (Thermo Fisher Scientifics) supplemented with 10 mM 130 HEPES, 50 µg/ml penicillin/streptomycin, 55 µM 2-mercaptoethanol (Thermo Fisher Scientifics), 131 and 10% heat-inactivated [56ºC, 30 min] fetal bovine serum (Atlanta Biologicals, Flowery Branch, 132 GA). 133 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 4 134 135 Plasmid construction 136 137 The recombinant protein vaccine that consists of the extracellular portions of EBOV GP lacking the 138 MLD (WT GPΔM) and a similar version that consists of BCG Th epitopes fused to the N terminus of 139 the EBOV GP (Th GPΔM) were constructed and described previously 33. The full-length EBOV GP 140 with the MLD restored was also constructed for these recombinant protein vaccines. To construct the 141 full-length version of the EBOV GP vaccines, the MLD of the EBOV GP was amplified from 142 pMAM01 40 using primer pair TN258 (5΄-143 GCGCACCGTCGTGTCAAACGGAGCCAAAAACATCAGTGG-3΄) and TN259 (5΄-144 GCGCCAGTATCCTGGTGGTGAGTGTTGTTGTTGCCAGCGG-3΄). The MLD was cloned into 145 WT GP-ΔM and Th GP ΔM via the AleI and XcmI sites to create the corresponding version WT GP-146 FL and Th GP-FL which contain the MLD in the EBOV GP. 147 148 149 Expression and purification of recombinant protein vaccines 150 151 Plasmids corresponding to WT GP-FL and Th GP-FL DNA were transfected into FreeStyle 293-F 152 cells, and proteins were collected from culture supernatants and purified using the HisTrap HP 153 column (GE Healthcare Life Sciences, Pittsburgh, PA) as described previously 33. Protein 154 concentrations were determined by the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). 155 The purified proteins (220ug/ml WT GP-GL and 80ug/ml Th GP-FL) in PBS were stored at -80ºC 156 until needed. Purified Th vaccines were analyzed by size-exclusion high performance liquid 157 chromatography (SE-HPLC) using the SRT SEC-300 size exclusion column. Analysis of the 158 molecular weight was determined by comparing the retention time with markers of known molecular 159 weight (BioRad). 160 161 162 SDS-PAGE analysis of recombinant protein vaccines 163 164 Purified recombinant protein vaccines were analyzed on SDS-PAGE by staining with GelCode Blue 165 Safe Protein Stain (Thermo Fisher Scientific). Proteins separated by SDS-PAGE were also 166 transferred onto nitrocellulose membranes for immunoblotting. After blocking with 5% bovine milk 167 in PBS with 0.05% Tween 20 (PBST), the nitrocellulose membranes containing the purified 168 recombinant fusion proteins were incubated with mouse anti-His antibody [clone HIS.H8] (Millipore 169 Sigma, Burlington, Massachusetts). HRP-conjugated rabbit anti-mouse IgG antibody 170 (SouthernBiotech, Birmingham, AL) were used as detection Abs, and signals were detected using the 171 SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). 172 173 174 T cell hybridoma stimulation assays 175 176 Mouse T cell hybridomas specific for peptide P25 of Ag85 or peptide P10 of TB9.8 were cocultured 177 with murine bone marrow-derived dendritic cells 33 and incubated with the purified recombinant 178 protein vaccine (10 µg/ml) at 37ºC for 18 h. Cell culture supernatants were assayed for IL-2 using 179 capture and biotin-labeled detection antibody pairs (BD Biosciences, Franklin Lakes, NJ). Detection 180 was performed with HRP-conjugated streptavidin (BD Biosciences) followed by the addition of the 181 Turbo 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific). 182 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 5 183 184 ELISA assays for anti-EBOV GP antibody titers 185 186 Measurement of anti-EBOV GP antibody titers was performed by direct solid phase ELISA. Corning 187 96-well flat bottom assay plate (Thermo Fisher Scientific) were coated with ~95,000 infectious unit 188 (IU) of recombinant vesicular stomatitis virus (rVSV) expressing EBOV GP (rVSV-EBOV) in PBS 189 (pH7.4) overnight at 4ºC 41,42. The EBOV GP coated ELISA plate was washed three times with PBS 190 and blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at RT. Serum samples from 191 immunized mice were obtained from blood collected by retro-orbital bleed in vaccinated mice. The 192 serum samples were diluted 1:50 in PBS for single dilution measurement or 1:20 followed by serial 193 1:3 dilutions for endpoint titers, and then incubated in the EBOV GP coated ELISA plate wells for 2 194 h at RT. The ELISA plates were then washed four times with PBS and incubated with HRP-195 conjugated mouse IgG1- or IgG2c-specific Abs (SouthernBiotech) for 1 h at RT. After washing four 196 times in PBS, the signal was detected with SIGMAFAST OPD substrate (Sigma-Aldrich, St. Louis, 197 MO) and the reaction was stopped with the addition of 0.5 M H2SO4. The absorbances for both the 198 capture and direct ELSIA assays were measured with the Wallac 1420 VICTOR2 microplate reader 199 (Perkin Elmer, Waltham, MA). 200 201 202 Flow cytometry analysis of Th subsets and FcR expression 203 204 Naïve CD4+ T cells from P25 TCR-Tg/GFP mice were purified by negative selection using a 205 commercially available kit and following the manufacturer’s instruction (Miltenyi Biotec, Auburn, 206 CA). During the CD4+ T cell purification, anti-CD44 conjugated to biotin [clone IM7] (Thermo 207 Fisher Scientific) was added in the purification step to remove memory T cells that were present in 208 these animals. 4 x 104 purified CD4+ T cells in 100 µl of PBS were injected intravenously via the tail 209 vein into WT C57BL/6 mice. Sixteen hours after injection of the CD4+ T cells, mice were vaccinated 210 with 100 µl of either 1 x 107 CFU of BCG in PBS or with 10 µg P25 peptide in PBS formulated with 211 one of the following adjuvants: 1:1 volume ratio of alum (Imject Alum; Thermo Fisher Scientific), or 212 5% final volume of LASTS-C [Span85-Tween 80-squalene, lipid A, CpG oligodeoxynucleotides] 213 43,44 (gift from Dr. Michael Anthony Moody, Duke University). On day 7 after vaccination, mice 214 were sacrificed, and spleens were harvested and cells were stained with Live Dead viability dye (LD 215 Fixable Blue; Thermo Fisher Scientific L34961) and antibodies against MHC class II (Alexa Fluor 216 700; BD 570802), CD4 (APC-Cy7, BD 561830), T-bet (PE-Cy7; Biolegend 644823), CXCR-5 (PE; 217 BD 551959), and Bcl-6 (APC; Biolegend 358505), and analyzed by FACS using the 5 laser BD 218 Biosciences LSRII Flow Cytometer, and 5 x 105 events per sample were collected and analyzed using 219 FlowJo software (BD biosciences). 220 For analysis of FcR expression, splenocytes from FcRII,III,IV -chains knockout mice 45 or 221 WT B6 vaccinated mice were processed at indicated timepoints and stained with mAbs against B220 222 (BUV661; BD 612972, clone: RA3-6B2), NK1.1 (BV605; BD 563220, clone: PK136), CD11c 223 (Alexa Fluor 700; BD 560583, clone: HL3), CD11b (PE-CF594; BD562287, clone: M1/70), Ly-224 6G/Ly-6C (APC; eBioscience 17-5931-81, clone: RB6-8C5), Ly6-C (PerCP; Biolegend 128028. 225 clone: HK1.4), FcRIV (PE; BD 565615, clone: 9E9), FcRII/III (FITC; BD 561726, clone: 2.4G2), 226 and analyzed by FACS using the Cytek Aurora configured with five lasers, three scattering channels, 227 and sixty-four fluorescence channels, and 1 x 106 events per sample were collected and analyzed 228 using the FlowJo software (BD biosciences). 229 230 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 6 231 Analysis of germinal centers 232 233 To detect the presence of antigen specific Th cells in the secondary lymphoid tissues, spleens from 234 vaccinated mice were sectioned and stained as described previously 33. Briefly, naïve CD4+ T cells 235 from P25 TCR-Tg/GFP mice were transferred into mice which were then vaccinated with either 1 x 236 107 BCG or with 10 µg P25 peptide formulated in LASTS-C as described in the analysis of Th 237 subsets above. On day 7 after vaccination, mice were sacrificed and spleens were fixed in 10% 238 neutral buffered formalin, paraffin embedded and sectioned. Tissue sections were stained with anti-239 GFP Ab (A11122; Thermo Fisher Scientific) for the presence of CD4+ T cells transferred from the 240 P25 TCR-Tg/GFP mouse and counterstained with hematoxylin. 241 242 243 Animal ethics statement 244 245 All infectious work with MA-EBOV was performed in the maximum containment laboratories at the 246 Rocky Mountain Laboratories (RML), Division of Intramural Research, National Institute of Allergy 247 and Infectious Diseases, National Institutes of Health. RML is an institution accredited by the 248 Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 249 All procedures followed standard operating procedures (SOPs) approved by the RML Institutional 250 Biosafety Committee (IBC). Mouse work was performed in strict accordance with the 251 recommendations described in the Guide for the Care and Use of Laboratory Animals of the National 252 Institute of Health, the Office of Animal Welfare and the Animal Welfare Act, United States 253 Department of Agriculture. The study was approved by the RML Animal Care and Use Committee 254 (ACUC). Procedures were conducted in mice anesthetized by trained personnel under the supervision 255 of veterinary staff. All efforts were made to ameliorate animal welfare and minimize animal 256 suffering; food and water were available ad libitum. 257 258 259 EBOV challenge in vaccinated mice 260 261 Wild-type female C57BL/6NHsd (approximately 10 weeks of age) were given 1 x 107 BCG in PBS 262 through s.c. injection at the scruff of the neck. Five weeks after exposure to BCG, the mice were 263 primed with 0.05 µg of the purified recombinant protein vaccine (WT GP-FL or Th GP-FL) 264 adjuvanted with alum in PBS through i.m. injection as described above. Vaccinated mice were rested 265 for 4 weeks, followed by a homologous boost of the recombinant protein vaccine administered 266 through the same route. Two weeks after each interval of administering the purified recombinant 267 protein vaccine, blood was collected through retro-orbital bleed to obtain serum samples for antibody 268 titer measurements. Four weeks after the boost, mice were shipped to Rocky Mountain Laboratories 269 in Hamilton, MT and rested for 1 week prior to MA-EBOV challenge. Mice were infected by 270 intraperitoneal (i.p.) injection of a lethal dose for naïve mice of 10 focus-forming units (FFU) of MA-271 EBOV 46. Five mice from each vaccinated group were euthanized on day 5 after challenge to harvest 272 organs to determine viremia and to collect blood samples to freeze down serum samples for future 273 analysis of anti-EBOV GP antibody responses. The remaining 10 mice from each vaccinated group 274 were kept under observation for survival and weight loss and all surviving mice were euthanized on 275 day 28 after challenge to collect and freeze serum samples. 276 277 278 ELISPOT to detect antigen specific T and B cells 279 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 7 280 For the T cell analysis, five-week-old female WT C57BL/6J mice (n = 10) were vaccinated by s.c. 281 injection of either PBS or BCG and rested for 5 weeks. Each group was further subdivided into 2 282 groups (n = 5) which received either PBS or the Th GP-FL vaccine at 0.05 μg per mouse in alum. 283 Splenocytes were obtained two weeks later for ELISPOT assay 47. Briefly, the 96-well ELISPOT 284 plate (Millipore) was prepared by coating the well with 50 μl of 10 μg/ml of anti-mouse IFN 285 monoclonal capture antibody (BD Biosciences; cat. no. 551309) in PBS and allowed to incubate at 286 4ºC for 16 hours. The ELISPOT wells were washed five times with PBST and blocked with 200 μg 287 of cRPMI for 2 hours at room temperature. Splenocytes at 5 x 105 cells per well were added along 288 with 5 μg/ml P25 peptide or 10 μg/ml of M. tuberculosis (strain H37Rv) lysate for antigen 289 stimulation and incubated at 37ºC in 5% CO2 for 16 hours. The ELISPOT plate was washed five 290 times with PBST and 50 μl of 1 μg/ml of the anti-mouse IFN monoclonal detection antibody 291 conjugated to biotin (BD Biosciences; cat. no. 551506) in PBS was added and allowed to incubate at 292 room temperature for 2 hours. The wells were then washed five times with PBS + 0.1% Tween-20 293 (PBST) and streptavidin-alkaline phosphatase (Thermo Fisher Scientific) at 1:1000 dilution in PBS 294 was added incubated at 37ºC in 5% CO2 for 1 hour. After a final 5 washes with PBST, the spots 295 were developed by adding the BCIP/NBT substrate (Sigma Aldrich). The reaction was stopped by 296 washing the wells with water and the spots were counted using an automated ELISPOT reader 297 (Autoimmun Diagnostika GmbH, Strasbourg, Germany). 298 For ELISPOT quantitation of antibody secreting cells 48, five-week-old female WT C57BL/6 mice 299 were vaccinated with PBS or BCG by s.c. administration of 1 x 107 CFU per mouse. Five weeks 300 after vaccination, mice were injected i.m. with 5 μg of the Th GP-FL vaccine adjuvanted with alum 301 in PBS. Thirty-nine weeks later, the mice were sacrificed to obtain the splenocytes and bone marrow 302 cells, which were immediately assayed by ELISPOT to detect antibody secreting B cells. The 96-303 well ELISPOT plate (Millipore) was prepared by coating the wells with 50 μl of 10 μg/ml of rVSV 304 expressing EBOV GP and incubating at 4ºC for 16 hours. The ELISPOT wells were washed five 305 times with PBS and blocked with 200 μl of cRPMI for 2 hours at room temperature. Splenocytes or 306 bone marrow cells at 106 cells per well were added to the ELISPOT plate and incubated at 37ºC in 307 5% CO2 for 5 hours. The ELISPOT plate was washed five times with PBS and anti-mouse IgG1 or 308 anti-mouse IgG2c antibodies conjugated with alkaline phosphatase (Southern Biotech) at 1:1000 in 309 PBS were added and incubated at room temperature for 2 hours. The spots were developed by 310 adding the BCIP/NBT substrate (Sigma Aldrich). The reaction was stopped by washing the wells 311 with water and the spots were counted using an automated ELISPOT reader (Autoimmun 312 Diagnostika GmbH, Strasbourg, Germany). 313 Comparative immunogenicity of EBOV GP vaccine constructs 314 315 Subunit vaccines against EBOV have shown potential for inducing protection against infection in 316 several animal models and may have important advantages over virally vectored vaccines 49. 317 However, optimal design of subunit vaccines regarding efficacy, potency, stability and formulation 318 issues requires further investigation and testing 50. The design of the EBOV glycoprotein (GP) Th-319 vaccine for the current study was based on our previous work showing the general impact of 320 incorporating immunodominant Th epitopes of BCG into a soluble version of EBOV GP from which 321 the mucin-like domain (MLD; EBOV GPMLD) was deleted to direct responses against conserved 322 epitopes important for neutralizing antibodies 33,40. Here we developed a new version of the EBOV 323 GP Th-vaccine that consisted of the full-length complete extracellular portion of the EBOV GP for 324 direct comparison with the previous version of EBOV GPMLD. Although the EBOV GPMLD 325 was produced with higher yields as a recombinant protein, the full-length version of the EBOV GP 326 Th-vaccine has the advantages of more closely resembling the native protein on the viral envelope or 327 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 8 surface of infected cells, and also could provide a greater range of epitopes for antibody targeting of 328 membrane expressed GP. As shown schematically (Fig. 1A), the immunodominant CD4+ T cell 329 epitopes of mycobacterial antigens Ag85B (P25 epitope) and TB9.8 (P10 epitope) were fused to the 330 N terminus of the full-length EBOV GP or to a version of EBOV GPMLD. These were designated 331 Th GP-FL or Th GP-ΔM, respectively. Protein expression was done in FreeStyle 293-F cells and 332 purified by Ni-NTA affinity chromatography as described previously 33. Versions of these proteins 333 lacking the N-terminal extension encoding the BCG Th epitopes, designated as wild type (WT), were 334 also constructed and purified to serve as controls. 335 Protein purity and quality were assessed by SDS-PAGE and immunoblotting. As shown by 336 Coomassie staining (Fig. 1B, left panel), the mature form (GP0) and the two proteolytic fragments 337 (GP1 and GP2) of the full-length (WT GP-FL and Th GP-FL) and the MLD versions (WT GP-ΔM 338 and Th GP-ΔM) of EBOV GP constructs were observed and confirmed to be of the expected sizes 339 40,51. As expected, immunoblotting with antibody specific for the hexahistidine tag at the carboxyl-340 terminal of the 25 kDa GP2 precursor (Fig. 1B, right panel) detected the mature form GP0 and the 341 GP2 cleavage product, but not the GP1 fragment which lacks the histidine tag. Since EBOV GP 342 exists mainly as trimers in its native cell surface form, we also carried out size exclusion 343 chromatography to analyze monomeric versus multimeric state of the soluble GP constructs in 344 solution 52,53 (Supplemental Fig. 1). This showed retention times consistent with mass of 600 kDa or 345 more for the proteins in solution, indicating complexes at least as large or larger than the expected 346 size for soluble trimers. This suggested that the subunit vaccines produced here were likely to be a 347 mixture of trimers and higher order multimers. 348 Consistent with the correctly folded structure for at least a fraction of the purified GP 349 preparations, the conformation sensitive anti-EBOV GP antibodies ADI-15878 and KZ52 54 bound to 350 all of the purified proteins in solid phase ELISA, (Fig. 1C and Supplemental Fig. 2). To demonstrate 351 correct processing and presentation of the BCG epitopes embedded in the Th (FL) and Th (ΔM) 352 fusion proteins for T cell recognition, we used previously isolated mouse T cell hybridomas specific 353 for the Ag85B or TB9.8 epitopes presented by MHC class II I-Ab molecules 33,55. T cell hybridoma 354 cells cultured with mouse bone marrow derived dendritic cells secreted IL-2 into the culture 355 supernatants in response to the purified GPs containing the Th sequence encoding the relevant T cell 356 epitopes, indicating efficient antigen processing at the inserted cathepsin S cleavage sites and 357 presentation by I-Ab (Fig. 1D). Furthermore, the BCG epitopes incorporated into the Th vaccines 358 were targeted by long-lived memory Th cells in BCG vaccinated mice. This was apparent in mice 359 vaccinated with PBS or BCG and then rested for 17 weeks before administrating the Th GP-FL 360 vaccine or PBS sham control. Two weeks later, IFN ELISPOT assays were performed on 361 splenocytes to determine recall responses of BCG specific Th cell against the peptide-25 (P25) of the 362 immunodominant Ag85B or Mtb (strain H37Rv) lysate (Figs. 2A and B). Mice in both of the BCG 363 vaccinated groups developed BCG specific Th cells reactive to Mtb lysate, but only the BCG group 364 that was subsequently immunized with the Th GP-FL vaccine showed significant expansion of P25 365 specific Th cells. 366 To test the immunogenicity of the full length Th GP-FL vaccine and compare this directly 367 with the MLD version (Th GPM) that we previously showed to lower the vaccine dose required to 368 induce antibody responses and induce IgG2c class switching 33, mice were vaccinated with BCG or 369 received sham vaccination with PBS injection, and then primed and boosted by subcutaneous 370 injections of either the Th GP-FL or Th GPM in alum. A solid phase ELISA was performed to 371 detect the presence of anti-EBOV GP-IgG1 and -IgG2c antibodies. Confirming our previously 372 published findings 33, the BCG-specific Th cells from prior BCG vaccination, which were absent in 373 the sham vaccinated (PBS) groups, were recruited by the Th vaccine to promote class switching to 374 IgG2c, and either version (Th GPM or Th GP-FL) of the Th vaccine induced similar antibody levels 375 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 9 (Fig. 2C). Taken together, these results showed that the Th GP-FL can be used to replace the Th 376 GPM version of the vaccine to more accurately represent the native form of GP associated with 377 actual EBOV infection and provide the broadest array of potential epitopes for both neutralizing and 378 non-neutralizing antibodies. 379 380 381 BCG vaccination upregulates FcRIV expression and supports long-lived antibody responses. 382 383 Non-neutralizing antibodies mediate their functions primarily through the binding of FcRs to recruit 384 immune cell effector functions, including cytolysis and phagocytosis, to clear infected cells. In mice, 385 the Fc portions of the IgG2 isotypes have the highest affinities for FcRIV, which is abundantly 386 expressed on monocytes, macrophages, and neutrophils 31, and to a lesser degree on NK cells 56,57. 387 To determine the expression level of FcRIV on immune cells, flow cytometry was performed to 388 identify B cells (B220+) NK cells (NK1.1+), neutrophils (CD11bhigh Ly6Ghigh), macrophages 389 (CD11bhigh Ly6Chigh) and monocytes (CD11bhigh Ly6Clow) (Fig. 3A). At 2 weeks after BCG 390 vaccination, an increase in the levels of FcRIV was detected on monocytes, macrophages, NK cells, 391 and B cells (Fig. 3B). Although the Th vaccine expanded the BCG memory Th cells (Fig. 2), this 392 was not associated with further enhancement of the BCG induced FcRIV expression at 17 weeks 393 after the initial BCG vaccination (Fig. 4A & B). However, these findings showed that BCG 394 vaccination induced prolonged elevation of FcRIV expression on effectors cells, which is likely to 395 be relevant to the efficacy of the Th vaccine design that favors the induction of IgG2c class-switched 396 antibodies 33. 397 To determine the duration of the persistence of antibodies against EBOV GP in mice 398 receiving the Th GP-FL vaccine, mice were either vaccinated with BCG or sham vaccinated (PBS 399 only), and then immunized with 5 μg of Th GP-FL (Fig. 5). In this experiment, a higher dose of the 400 Th GP-FL was given to the animal instead of the usual dose of 0.5 μg per mouse in order to elicit a 401 detectable IgG2c response in the PBS group for comparison with the BCG group. Serum samples 402 were collected at times ranging from 2 to 39 weeks after the administration of the Th GP-FL vaccine 403 and analyzed by ELISA for anti-EBOV GP titers for both IgG1 and IgG2c subclasses. Compared to 404 the PBS group that lacked BCG specific Th1 cells, the intrastructural help provided by BCG specific 405 Th1 cells in the BCG vaccinated group promoted higher anti-EBOV GP titers. Anti-EBOV GP 406 antibodies were detected even at week 39 after vaccination (Fig. 5A), and, at the same time, plasma 407 cells secreting these anti-EBOV GP antibodies were detected in bone marrow and not the spleen (Fig. 408 5B), indicating that long lived plasma cells induced by the Th vaccine can elicit long lasting 409 protection. 410 411 412 BCG vaccination induced extrafollicular Th1 responses and altered germinal center formation. 413 414 In our previous publication, we showed that antibodies induced by the Th vaccine have different 415 affinities in the Fab region that correlated with IgG1 and IgG2c isotypes 33. B cells that enter the 416 germinal center (GC) form cognate interaction with T follicular helper (Tfh) cells, which are defined 417 by expression of CXCR5 and the lineage-defining transcription factor Bcl-6 58, and go through 418 multiple rounds of affinity maturation to develop high affinity antibodies. B cells that encounter 419 antigens outside of GCs undergo cognate interactions with non-Tfh cells such as Th1 cells, which 420 reduces affinity maturation but provides rapid protection in early stages of infection 59. To visualize 421 the location of BCG-specific Th cells within a secondary lymphoid organ after BCG vaccination, we 422 used adoptive transfer of GFP labelled CD4+ T cells expressing a TCR transgene specific for the P25 423 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 10 epitope of BCG Ag85B as previously described 33. To compare the extent to which these adoptively 424 transferred T cells remained extrafollicular or were capable of entering germinal centers, we 425 compared mice vaccinated with BCG versus mice immunized with P25 peptide combined with 426 various adjuvants, including alum and a multicomponent formulation known as LASTS-C (lipid A, 427 Span8, Tween 80 and CpG oligodeoxynucleotides) 43. 16 hours after immunization, splenocytes 428 were isolated and analyzed by FACS with gating on CD4+ GFP+ cells (Fig. 6A; top panel). Th1 429 polarization of the transferred P25-specific GFP+ CD4+ T cells as determined by Tbet expression was 430 strongest in BCG vaccination as compared with other adjuvants (Fig. 6A; middle panel), whereas the 431 Tfh polarization as shown by CXCR5 and Bcl-6 double staining was extremely low except in 432 animals receiving vaccination with the LASTS-C adjuvant (Fig. 6A; lower panel), which correlates 433 with its ability to induce strong neutralizing antibody responses 44. 434 To further evaluate the effects of BCG vaccination on the functional outcomes of CD4+ T cell 435 responses, we analyzed the localization of P25 specific T cells in the spleen by 436 immunohistochemistry. Naïve P25-specific GFP+ CD4+ T cells were transferred intravenously into 437 mice that were vaccinated 16 hours later with either BCG or the P25 peptide adjuvanted in LASTS-438 C. Six days after vaccination, spleens were isolated, sectioned, and analyzed by 439 immunohistochemistry with anti-GFP staining followed by H&E counter staining (Fig. 6B). BCG 440 vaccination, as expected for strong Th1 biasing stimuli, diminished the formation of GCs (Fig. 6B; 441 left panel) as compared to non-Th1 adjuvant such as LASTS-C (Fig. 6B; right panel), which was 442 quantified by counting the number of GC per follicle (Fig. 6B). In BCG vaccinated mice, the GFP+ 443 CD4+ T cells, observed as brown precipitate of the 3,3-diaminobenzidine in the 444 immunohistochemistry staining with anti-GFP conjugated with horse radish peroxidase, were present 445 in the white pulp areas (Fig. 6B; left panel) but nearly absent in the relatively scarce GCs (Fig. 6C; 446 left enlarged panel). In contrast, in the LASTS-C vaccination group (Fig. 6B; right panel) there were 447 numerous GCs, and the GFP+ CD4+ T cells were mostly localized within the GCs (Fig. 6C; right 448 enlarged panel), as quantified as the number of P25 cells per GC (Fig. 6C). These data suggested 449 that the majority of BCG Th cells remained outside the GC at the border of the B cell follicle and 450 likely exerted their effects on the antibody response at this location comprising the boundary of B 451 and T cell zones. 452 453 454 Vaccination with subunit vaccine for protection against EBOV challenge. 455 456 Experiments to assess our proposed regimen for protection from lethal EBOV infection required the 457 use of mice that will have reached 24 weeks of age at the time of infection with EBOV, an age group 458 that has not to our knowledge been previously tested in the EBOV challenge model. To determine 459 whether mice at this age have similar susceptibility to EBOV infection compared to the typically 460 used younger mice, 24-week-old mice were challenged with 10 or 1,000 focus-forming units (FFU) 461 of mouse-adapted EBOV (MA-EBOV). Eight days after challenge, 24-week-old mice succumbed to 462 the MA-EBOV infection even with the lower 10 FFU infectious dose (Supplemental Fig. 3), which 463 was similar to the time to death observed previously in younger mice at 6-14 weeks of age 46. 464 Having established the susceptibility of the older mice in this model, we tested the efficacy of our 465 regimen using recombinant protein vaccines augmented by prior BCG vaccination to protect against 466 EBOV challenge using the vaccination strategy illustrated in Figure 7A. Sera were analyzed by 467 ELISA for anti-EBOV GP antibody responses 2 weeks after priming and again after boosting with 468 the subunit vaccines. The group receiving the WT GP-FL vaccine (WT), which is incapable of 469 recruiting BCG Th cells for intrastructural help, failed to induce robust anti-EBOV GP antibody 470 responses, and did not undergo IgG2c class-switching (Fig. 7B, left). This was in contrast to the Th 471 GP-FL vaccine (Th), which enhanced anti-EBOV GP antibody responses and IgG2c class-switching 472 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 11 (Fig. 7B, right). An ELISA performed with end-point dilution of the serum samples collected after 473 the Th-vaccine boost showed similar results, with the Th GP-FL vaccine providing elevated levels of 474 both IgG1 and IgG2c antibodies against EBOV GP (Fig. 7C). Five weeks after boosting with the 475 subunit vaccines, mice were challenged with 10 FFU of MA-EBOV. All the mice from the PBS and 476 the WT GP-FL vaccine group succumbed to the infection 8 days after MA-EBOV challenge, whereas 477 the majority of the mice in the Th GP-FL vaccine group survived through the end of the study, at 478 which point they appeared healthy and had regained their original body weight (Fig. 7D). In 479 addition, at day 5 after EBOV infection, five mice were sacrificed to determine the viral titers in the 480 blood and organs. Mice that received the Th GP-FL vaccine had a lower viral titer in the blood, liver, 481 and spleen when compared to mice that either received no vaccination or the WT GP-FL (Fig. 7E). 482 An ELISA was also performed on the serum samples collected from these mice which showed that 483 mice that received the Th GP-FL vaccine also had a higher anti-EBOV GP antibody titer compared to 484 the control group receiving PBS only or the WT GP-FL vaccine group (Fig. 7F). Long term 485 survivors from the Th vaccine group were also bleed at termination of the experiment (day 112, 486 corresponding to 14 days after EBOV challenge), and analysis of these serum samples showed 487 persistently high levels of anti-EBOV GP antibodies (Fig. 7G). Thus, the Th vaccine strategy clearly 488 protected the mice against lethal EBOV infection by limiting viral replication to control the early 489 stage of infection, which is known to be important in conferring protection as seen in other viral 490 infections 59. 491 492 493 The approach to antiviral vaccination used in the current study is based on the classic hapten-carrier 494 immunization studies that led to the understanding of the concept of linked recognition. Many 495 effective vaccines depend on the core immunological concept of linked-recognition, in which Th 496 cells recognize processed peptides derived from the immunogen targeting B cell receptors to provide 497 intrastructural help to B cells, leading to T cell dependent antibody responses 60. These include 498 vaccines that rely on the production of antibodies against targets that entirely lack T cell epitopes, 499 such as those against Haemophilus influenzae type b (Hib) polysaccharides 61 or small hapten-like 500 molecules like nicotine 62. In these cases, conjugation to a protein carrier containing Th cell epitopes 501 is required to elicit an optimal T cell dependent B cell response. In a logical extension of this 502 principle, we and others have applied this approach to creating protein subunit vaccines against viral 503 antigens to enhance and accelerate protective antibody responses through the recruitment of pre-504 existing Th cells against other potent antigens, such as those delivered by previous vaccination 505 against pathogens such as mycobacteria. For example, Klessing et al. developed a vaccine against 506 HIV that can recruit intrastructural help from Th cells induced by an M. tuberculosis subunit vaccine, 507 and showed that this approach induced higher antibody titers that persisted for extended period of 508 time 63. In our previous work we applied a similar approach to capture intrastructural help to B cells 509 from pre-existing Th1 cells specific for immunodominant mycobacterial antigens in BCG vaccinated 510 mice 33. In the current study, we expanded on our previous work to determine the protective efficacy 511 of this Th vaccine design against EBOV challenge in the mouse model, and to explore in greater 512 detail the potential mechanisms mediating this protection. Consistent with our findings, we showed 513 that this vaccine strategy induced antibody class-switching to IgG2c, an isotype that is known to have 514 high affinity toward FcRIV, suggesting that antibodies with effector activities such as antibody-515 mediated cellular cytotoxicity (ADCC) might be a key feature that extended the antiviral effects 516 beyond simple neutralization of viral entry. 517 Our previous efforts to generate fusion proteins for use as subunit vaccines against EBOV 518 used a truncated form of EBOV GP in which the MLD was deleted, which our preliminary work had 519 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 12 shown to be produced with much higher yields than a full-length version (Th GP-FL) that retains the 520 MLD 33. In the current study, we further improved production and purification of the Th GP-FL 521 fusion protein and formulated this with alum to generate a candidate vaccine against EBOV infection 522 and disease for use in previously BCG vaccinated hosts. This full-length version of the extracellular 523 domains of EBOV GP should present an immunogen that corresponds more closely than the 524 previously designed Th GPM vaccine that lacks the MLD to the actual infectious virus or the form 525 of the EBOV GP expressed on the surface of infected host cells, making it potentially more effective 526 for generating a broad range of antibodies mediating a variety of host protective functions. In this 527 regard, while epitopes in the MLD region have not been strongly associated with broad neutralization 528 of viral entry, such antibodies may play an important role in controlling the progression and spread of 529 infection through non-neutralizing activities such as ADCC 64. 530 Our preparations of the Th GP-FL fusion protein produced so far appeared to exist mainly as 531 higher order multimers in solution, rather than as soluble monomers or native homotrimers 532 (Supplemental Fig. 1). The multimerization of the protein may be due to artifactual disulfide bond 533 formation or other tight interactions that formed during the purification, and suggests the need for 534 further optimization of the production and purification process. However, irrespective of the 535 presence of larger multimeric complexes in the Th GP-FL preparations, the native conformation of 536 the protein appeared to be present at significant levels, as shown by its recognition by the anti-EBOV 537 GP monoclonal antibodies, ADI-15878 and KZ52, which recognize conformational epitopes of the 538 native protein (Fig. 1C and Supplemental Fig. 2). Furthermore, the Th GP-FL vaccine was able to 539 induce anti-EBOV GP antibodies that recognized EBOV GP expressed on the surface of recombinant 540 vesicular stomatitis virus (Figs. 2C, 5A, and 7B). Most importantly, the vaccine conferred protection 541 against challenge with MA-EBOV (Fig. 7D & E), indicating that antibodies generated by the Th GP-542 FL vaccine, particularly when administered in the context of prior BCG vaccination, were able to 543 recognize the relevant form of GP during viral infection. 544 A key feature of our vaccine strategy is the prior BCG vaccination, which not only induced 545 memory BCG-specific Th1 cells to provide intrastructural help, but also through trained immunity, 546 can enhance non-specific immune mechanisms 65. Protection from trained immunity induced by 547 BCG has been described in COVID-19 infection and also in BCG-based bladder cancer treatments 66. 548 In our analyses, we observed that BCG exposure also promoted FcRIV expression (Figs. 3 and 4) 549 and IgG2c class-switching (Fig. 2C), which can be viewed as additional aspects of trained immunity. 550 In the mouse model, the IgG2c isotype and FcRIV expression together are important for the 551 induction of ADCC by certain immune effector cells such as neutrophils and NK cells. Correlating 552 with the induction of anti-EBOV GP IgG2c antibodies together with FcRIV expression, BCG-553 vaccinated mice that received the Th GP-FL vaccine, but not those with the WT GP-FL vaccine, 554 survived the EBOV challenge (Fig. 7D). This suggests that the anti-EBOV GP IgG2c isotype (Fig. 555 7B) played a significant role in conferring this protection. Analyzing the location of the BCG Th1 556 cells revealed that the anti-EBOV GP IgG2c antibodies were likely derived from extrafollicular 557 plasmablasts since BCG vaccination induces a strong Th1 cell response that favor less GC 558 development as compared to a Tfh promoting adjuvant (Fig. 6). As a result of this massive Th1 559 polarization, most of the T-dependent B cells are activated by the Th1 cells at the boundary of B cell 560 follicles and not inside GCs. These GC-nonresident B cells develop into extrafollicular plasmablasts 561 which are usually short-lived. Surprisingly, at 39-weeks after vaccination anti-EBOV GP antibody 562 levels were still detected in vaccinated mice (Fig. 5A) and the EBOV GP-specific antibody secreting 563 cells were still detected in the bone marrow (Fig. 5B), presumably representing long-lived plasma 564 cells. This suggests the possibility that GC in BCG vaccinated mice, although not initially detected, 565 may form at a later time and enable extrafollicular B cells induced in early stages post-vaccination to 566 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 13 develop into memory B cells and long-lived plasma cells that take up residence in the bone marrow 567 to sustain long term production of anti-EBOV GP antibodies. 568 Although other forms of EBOV vaccine are available such as the VSV-based EBOV vaccine 569 (Ervebro) that is FDA approved for human use 67-69 and confers protection in non-human primates 10 570 days after vaccination 70, the disadvantages faced by virus vectored EBOV vaccines are 571 manufacturing difficulties for large scale production and cold chain requirement during distribution 572 69, which can easily overwhelm logistics when dealing with larger outbreaks. Other EBOV vaccine 573 platforms require strong adjuvants to increase immunogenicity 49,50,71, including recombinant subunit 574 vaccines based on EBOV GP that are currently being developed 49,50,71. The development of a 575 subunit EBOV vaccine should allow easier production and distribution, especially in resource limited 576 nations, which can contribute to rapid deployment to control outbreaks 72. The recombinant protein 577 vaccine used in this study has the unique ability to recruit BCG-specific Th1 cells to provide 578 intrastructural help for driving antibody production against the recombinant protein subunit without 579 the use a strong adjuvant that can increase cost and unwanted side effects. These properties can also 580 lower the dose of the recombinant vaccine required, which can have an impact on manufacturing, 581 cost, and distribution worldwide. Furthermore, sustained antibody responses through week 39 was 582 observed after administering a single dose of the Th GP-FL vaccine. The benefit of the Th GP-FL 583 vaccine developed in this study as a recombinant protein, no doubt, is its simplicity as compared to a 584 virus vaccine, and its ability to harness pre-existing BCG-induced immunity to protect mouse against 585 MA-EBOV infection. Based on current projections 73, BCG vaccination will continue in many 586 regions of the world well into the future 74, thus establishing large populations that should be well 587 suited for mass vaccination against EBOV or other emerging viruses 75 using the approach 588 demonstrated by the current study. 589 590 5 Conflict of Interest 591 The authors declare that the research was conducted in the absence of any commercial or financial 592 relationships that could be construed as a potential conflict of interest. 593 594 6 Author Contributions 595 TWN and SAP: experimental conception, design, analysis, interpretation of data, and writing of the 596 manuscript. TWN: performed experiments and the analysis and acquisition of data. WF and AM: 597 design, performed, acquired, and analyzed data for the EBOV mouse challenge. ASW: prepared the 598 rVSV EBOV GP. NASA: assisted with analysis of histology data of spleen sections. CTJ: assisted 599 with the FACS analyses. AM, WRJ, and KC: analyzed, interpreted experiments, and reviewed the 600 manuscript. All authors critically reviewed and approved the manuscript. 601 602 7 Funding 603 The core facilities used in this study were all supported in part by NCI Cancer Center Service Grant 604 P30CA013330. Shared instrumentation grants funded the purchase of the Cytek Aurora FACS 605 analyzer (S10OD026833-01) and the 3DHistec Panoramic 250 Flash II slide scanner 606 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 14 (1S10OD019961-01) used in this study. This study was in part supported by the Intramural Research 607 Program, NIAID, NIH (AM). 608 609 8 Acknowledgments 610 Resources and advice were provided by core facilities at Albert Einstein College of Medicine, 611 including the Flow Cytometry, Analytic Imaging and Histopathology facilities. We thank Dr, Scott 612 Garforth and the Macromolecular Therapeutics Development Facility at Albert Einstein College of 613 Medicine for performing the size exclusion chromatography. We also thank Mei Chen and John Kim 614 (Department of Microbiology & Immunology, Albert Einstein College of Medicine) for expert 615 technical assistance with mouse experiments. We thank Bing Chen (Department of Microbiology & 616 Immunology, Albert Einstein College of Medicine) for assistance in maintaining of FcR KO mice 617 colony. We also thank the animal care takers of the Rocky Mountain Veterinary Branch (NIAID, 618 NIH) for their support of the EBOV mouse challenge. 619 620 9 References 621 1 Jones, K. E. et al. Global trends in emerging infectious diseases. Nature 451, 990-993 (2008). 622 https://doi.org:10.1038/nature06536 623 2 Mohammed, I. et al. The efficacy and effectiveness of the COVID-19 vaccines in reducing 624 infection, severity, hospitalization, and mortality: a systematic review. Hum Vaccin 625 Immunother 18, 2027160 (2022). https://doi.org:10.1080/21645515.2022.2027160 626 3 Cohen, Y. Z. & Caskey, M. Broadly neutralizing antibodies for treatment and prevention of 627 HIV-1 infection. Curr Opin HIV AIDS 13, 366-373 (2018). 628 https://doi.org:10.1097/COH.0000000000000475 629 4 Haynes, B. F. & Mascola, J. R. The quest for an antibody-based HIV vaccine. Immunol Rev 630 275, 5-10 (2017). https://doi.org:10.1111/imr.12517 631 5 Nishimura, Y. & Martin, M. A. Of Mice, Macaques, and Men: Broadly Neutralizing 632 Antibody Immunotherapy for HIV-1. Cell Host Microbe 22, 207-216 (2017). 633 https://doi.org:10.1016/j.chom.2017.07.010 634 6 Mielke, D. et al. ADCC-mediating non-neutralizing antibodies can exert immune pressure in 635 early HIV-1 infection. PLoS Pathog 17, e1010046 (2021). 636 https://doi.org:10.1371/journal.ppat.1010046 637 7 Pinto, D. et al. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV 638 antibody. Nature 583, 290-295 (2020). https://doi.org:10.1038/s41586-020-2349-y 639 8 Yu, Y. et al. Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-640 19 patients. Signal Transduct Target Ther 6, 346 (2021). https://doi.org:10.1038/s41392-021-641 00759-1 642 9 Gorander, S., Ekblad, M., Bergstrom, T. & Liljeqvist, J. A. Anti-glycoprotein g antibodies of 643 herpes simplex virus 2 contribute to complete protection after vaccination in mice and induce 644 antibody-dependent cellular cytotoxicity and complement-mediated cytolysis. Viruses 6, 645 4358-4372 (2014). https://doi.org:10.3390/v6114358 646 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 15 10 Petro, C. D. et al. HSV-2 DeltagD elicits FcgammaR-effector antibodies that protect against 647 clinical isolates. JCI Insight 1 (2016). https://doi.org:10.1172/jci.insight.88529 648 11 Bonsignori, M. et al. Antibody-dependent cellular cytotoxicity-mediating antibodies from an 649 HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene 650 family. J Virol 86, 11521-11532 (2012). https://doi.org:10.1128/JVI.01023-12 651 12 Karnasuta, C. et al. Antibody-dependent cell-mediated cytotoxic responses in participants 652 enrolled in a phase I/II ALVAC-HIV/AIDSVAX B/E prime-boost HIV-1 vaccine trial in 653 Thailand. Vaccine 23, 2522-2529 (2005). https://doi.org:10.1016/j.vaccine.2004.10.028 654 13 Kim, J. H., Excler, J. L. & Michael, N. L. Lessons from the RV144 Thai phase III HIV-1 655 vaccine trial and the search for correlates of protection. Annu Rev Med 66, 423-437 (2015). 656 https://doi.org:10.1146/annurev-med-052912-123749 657 14 Koch, T. et al. Ebola Virus Disease Survivors Show More Efficient Antibody Immunity than 658 Vaccinees Despite Similar Levels of Circulating Immunoglobulins. Viruses 12 (2020). 659 https://doi.org:10.3390/v12090915 660 15 Wren, L. H., Stratov, I., Kent, S. J. & Parsons, M. S. Obstacles to ideal anti-HIV antibody-661 dependent cellular cytotoxicity responses. Vaccine 31, 5506-5517 (2013). 662 https://doi.org:10.1016/j.vaccine.2013.08.035 663 16 Paquin-Proulx, D. et al. Associations Between Antibody Fc-Mediated Effector Functions and 664 Long-Term Sequelae in Ebola Virus Survivors. Front Immunol 12, 682120 (2021). 665 https://doi.org:10.3389/fimmu.2021.682120 666 17 Wagstaffe, H. R. et al. Antibody-Dependent Natural Killer Cell Activation After Ebola 667 Vaccination. J Infect Dis 223, 1171-1182 (2021). https://doi.org:10.1093/infdis/jiz657 668 18 Alpert, M. D. et al. ADCC develops over time during persistent infection with live-attenuated 669 SIV and is associated with complete protection against SIV(mac)251 challenge. PLoS Pathog 670 8, e1002890 (2012). https://doi.org:10.1371/journal.ppat.1002890 671 19 Worley, M. J. et al. Neutrophils mediate HIV-specific antibody-dependent phagocytosis and 672 ADCC. J Immunol Methods 457, 41-52 (2018). https://doi.org:10.1016/j.jim.2018.03.007 673 20 Doepker, L. E. et al. Development of antibody-dependent cell cytotoxicity function in HIV-1 674 antibodies. Elife 10 (2021). https://doi.org:10.7554/eLife.63444 675 21 Gunn, B. M. et al. A Fc engineering approach to define functional humoral correlates of 676 immunity against Ebola virus. Immunity 54, 815-828 e815 (2021). 677 https://doi.org:10.1016/j.immuni.2021.03.009 678 22 Gatto, D. & Brink, R. The germinal center reaction. J Allergy Clin Immunol 126, 898-907; 679 quiz 908-899 (2010). https://doi.org:10.1016/j.jaci.2010.09.007 680 23 Roco, J. A. et al. Class-Switch Recombination Occurs Infrequently in Germinal Centers. 681 Immunity 51, 337-350 e337 (2019). https://doi.org:10.1016/j.immuni.2019.07.001 682 24 Chen, Z. & Wang, J. H. Signaling control of antibody isotype switching. Adv Immunol 141, 683 105-164 (2019). https://doi.org:10.1016/bs.ai.2019.01.001 684 25 Zhang, Z., Goldschmidt, T. & Salter, H. Possible allelic structure of IgG2a and IgG2c in 685 mice. Mol Immunol 50, 169-171 (2012). https://doi.org:10.1016/j.molimm.2011.11.006 686 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 16 26 Barr, T. A., Brown, S., Mastroeni, P. & Gray, D. B cell intrinsic MyD88 signals drive IFN-687 gamma production from T cells and control switching to IgG2c. J Immunol 183, 1005-1012 688 (2009). https://doi.org:10.4049/jimmunol.0803706 689 27 Snapper, C. M. & Paul, W. E. Interferon-gamma and B cell stimulatory factor-1 reciprocally 690 regulate Ig isotype production. Science 236, 944-947 (1987). 691 https://doi.org:10.1126/science.3107127 692 28 Kuhn, R., Rajewsky, K. & Muller, W. Generation and analysis of interleukin-4 deficient 693 mice. Science 254, 707-710 (1991). https://doi.org:10.1126/science.1948049 694 29 Lee, F. et al. Isolation and characterization of a mouse interleukin cDNA clone that expresses 695 B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities. Proc 696 Natl Acad Sci U S A 83, 2061-2065 (1986). https://doi.org:10.1073/pnas.83.7.2061 697 30 Lutzker, S., Rothman, P., Pollock, R., Coffman, R. & Alt, F. W. Mitogen- and IL-4-regulated 698 expression of germ-line Ig gamma 2b transcripts: evidence for directed heavy chain class 699 switching. Cell 53, 177-184 (1988). https://doi.org:10.1016/0092-8674(88)90379-0 700 31 Bruhns, P. & Jonsson, F. Mouse and human FcR effector functions. Immunol Rev 268, 25-51 701 (2015). https://doi.org:10.1111/imr.12350 702 32 Nimmerjahn, F., Bruhns, P., Horiuchi, K. & Ravetch, J. V. FcgammaRIV: a novel FcR with 703 distinct IgG subclass specificity. Immunity 23, 41-51 (2005). 704 https://doi.org:10.1016/j.immuni.2005.05.010 705 33 Ng, T. W. et al. Exploiting Pre-Existing CD4(+) T Cell Help from Bacille Calmette-Guerin 706 Vaccination to Improve Antiviral Antibody Responses. J Immunol 205, 425-437 (2020). 707 https://doi.org:10.4049/jimmunol.2000191 708 34 Elsayed, H. et al. Intrastructural Help: Harnessing T Helper Cells Induced by Licensed 709 Vaccines for Improvement of HIV Env Antibody Responses to Virus-Like Particle Vaccines. 710 J Virol 92 (2018). https://doi.org:10.1128/JVI.00141-18 711 35 Nabi, G. et al. GagPol-specific CD4(+) T-cells increase the antibody response to Env by 712 intrastructural help. Retrovirology 10, 117 (2013). https://doi.org:10.1186/1742-4690-10-117 713 36 Kaneko, N. et al. Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in 714 COVID-19. Cell 183, 143-157 e113 (2020). https://doi.org:10.1016/j.cell.2020.08.025 715 37 Popescu, M., Cabrera-Martinez, B. & Winslow, G. M. TNF-alpha Contributes to Lymphoid 716 Tissue Disorganization and Germinal Center B Cell Suppression during Intracellular Bacterial 717 Infection. J Immunol 203, 2415-2424 (2019). https://doi.org:10.4049/jimmunol.1900484 718 38 Racine, R. et al. Impaired germinal center responses and suppression of local IgG production 719 during intracellular bacterial infection. J Immunol 184, 5085-5093 (2010). 720 https://doi.org:10.4049/jimmunol.0902710 721 39 Ryg-Cornejo, V. et al. Severe Malaria Infections Impair Germinal Center Responses by 722 Inhibiting T Follicular Helper Cell Differentiation. Cell Rep 14, 68-81 (2016). 723 https://doi.org:10.1016/j.celrep.2015.12.006 724 40 Martinez, O., Tantral, L., Mulherkar, N., Chandran, K. & Basler, C. F. Impact of Ebola 725 mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like 726 particles. J Infect Dis 204 Suppl 3, S825-832 (2011). https://doi.org:10.1093/infdis/jir295 727 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 17 41 Miller, E. H. et al. Ebola virus entry requires the host-programmed recognition of an 728 intracellular receptor. EMBO J 31, 1947-1960 (2012). https://doi.org:10.1038/emboj.2012.53 729 42 Wong, A. C., Sandesara, R. G., Mulherkar, N., Whelan, S. P. & Chandran, K. A forward 730 genetic strategy reveals destabilizing mutations in the Ebolavirus glycoprotein that alter its 731 protease dependence during cell entry. J Virol 84, 163-175 (2010). 732 https://doi.org:10.1128/JVI.01832-09 733 43 PCT/US2013/029164 - Vaccine Formulation. Moody, M. Anthony. (2013). 734 44 Moody, M. A. et al. Toll-like receptor 7/8 (TLR7/8) and TLR9 agonists cooperate to enhance 735 HIV-1 envelope antibody responses in rhesus macaques. J Virol 88, 3329-3339 (2014). 736 https://doi.org:10.1128/JVI.03309-13 737 45 Smith, P., DiLillo, D. J., Bournazos, S., Li, F. & Ravetch, J. V. Mouse model recapitulating 738 human Fcgamma receptor structural and functional diversity. Proc Natl Acad Sci U S A 109, 739 6181-6186 (2012). https://doi.org:10.1073/pnas.1203954109 740 46 Haddock, E., Feldmann, H. & Marzi, A. Ebola Virus Infection in Commonly Used 741 Laboratory Mouse Strains. J Infect Dis 218, S453-S457 (2018). 742 https://doi.org:10.1093/infdis/jiy208 743 47 Johnson, A. J., Kennedy, S. C., Ng, T. W. & Porcelli, S. A. Identification of Novel 744 Mycobacterial Targets for Murine CD4(+) T-Cells by IFNgamma ELISPOT. Methods Mol 745 Biol 1808, 143-150 (2018). https://doi.org:10.1007/978-1-4939-8567-8_12 746 48 Shah, H. B. & Koelsch, K. A. B-Cell ELISPOT: For the Identification of Antigen-Specific 747 Antibody-Secreting Cells. Methods Mol Biol 1312, 419-426 (2015). 748 https://doi.org:10.1007/978-1-4939-2694-7_42 749 49 Lehrer, A. T. et al. Recombinant Protein Filovirus Vaccines Protect Cynomolgus Macaques 750 From Ebola, Sudan, and Marburg Viruses. Front Immunol 12, 703986 (2021). 751 https://doi.org:10.3389/fimmu.2021.703986 752 50 Lehrer, A. T. et al. Recombinant subunit vaccines protect guinea pigs from lethal Ebola virus 753 challenge. Vaccine 37, 6942-6950 (2019). https://doi.org:10.1016/j.vaccine.2019.06.035 754 51 Volchkov, V. E., Feldmann, H., Volchkova, V. A. & Klenk, H. D. Processing of the Ebola 755 virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U S A 95, 5762-756 5767 (1998). https://doi.org:10.1073/pnas.95.10.5762 757 52 Hood, C. L. et al. Biochemical and structural characterization of cathepsin L-processed Ebola 758 virus glycoprotein: implications for viral entry and immunogenicity. J Virol 84, 2972-2982 759 (2010). https://doi.org:10.1128/JVI.02151-09 760 53 Rutten, L. et al. Structure-Based Design of Prefusion-Stabilized Filovirus Glycoprotein 761 Trimers. Cell Rep 30, 4540-4550 e4543 (2020). https://doi.org:10.1016/j.celrep.2020.03.025 762 54 Wec, A. Z. et al. Antibodies from a Human Survivor Define Sites of Vulnerability for Broad 763 Protection against Ebolaviruses. Cell 169, 878-890 e815 (2017). 764 https://doi.org:10.1016/j.cell.2017.04.037 765 55 Khan, A. et al. An autophagy-inducing and TLR-2 activating BCG vaccine induces a robust 766 protection against tuberculosis in mice. NPJ Vaccines 4, 34 (2019). 767 https://doi.org:10.1038/s41541-019-0122-8 768 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 18 56 Nimmerjahn, F. & Ravetch, J. V. Fcgamma receptors as regulators of immune responses. Nat 769 Rev Immunol 8, 34-47 (2008). https://doi.org:10.1038/nri2206 770 57 Sanseviero, E. et al. Anti-CTLA-4 Activates Intratumoral NK Cells and Combined with 771 IL15/IL15Ralpha Complexes Enhances Tumor Control. Cancer Immunol Res 7, 1371-1380 772 (2019). https://doi.org:10.1158/2326-6066.CIR-18-0386 773 58 Laurent, C., Fazilleau, N. & Brousset, P. A novel subset of T-helper cells: follicular T-helper 774 cells and their markers. Haematologica 95, 356-358 (2010). 775 https://doi.org:10.3324/haematol.2009.019133 776 59 Miyauchi, K. et al. Protective neutralizing influenza antibody response in the absence of T 777 follicular helper cells. Nat Immunol 17, 1447-1458 (2016). https://doi.org:10.1038/ni.3563 778 60 Ploegh, H. L. Bridging B cell and T cell recognition of antigen. J Immunol 179, 7193 (2007). 779 https://doi.org:10.4049/jimmunol.179.11.7193 780 61 Kelly, D. F., Moxon, E. R. & Pollard, A. J. Haemophilus influenzae type b conjugate 781 vaccines. Immunology 113, 163-174 (2004). https://doi.org:10.1111/j.1365-782 2567.2004.01971.x 783 62 Pentel, P. R. & LeSage, M. G. New directions in nicotine vaccine design and use. Adv 784 Pharmacol 69, 553-580 (2014). https://doi.org:10.1016/B978-0-12-420118-7.00014-7 785 63 Klessing, S. et al. CD4(+) T Cells Induced by Tuberculosis Subunit Vaccine H1 Can Improve 786 the HIV-1 Env Humoral Response by Intrastructural Help. Vaccines (Basel) 8 (2020). 787 https://doi.org:10.3390/vaccines8040604 788 64 Ilinykh, P. A. et al. Non-neutralizing Antibodies from a Marburg Infection Survivor Mediate 789 Protection by Fc-Effector Functions and by Enhancing Efficacy of Other Antibodies. Cell 790 Host Microbe 27, 976-991 e911 (2020). https://doi.org:10.1016/j.chom.2020.03.025 791 65 Kleinnijenhuis, J. et al. Long-lasting effects of BCG vaccination on both heterologous 792 Th1/Th17 responses and innate trained immunity. J Innate Immun 6, 152-158 (2014). 793 https://doi.org:10.1159/000355628 794 66 van Puffelen, J. H. et al. Trained immunity as a molecular mechanism for BCG 795 immunotherapy in bladder cancer. Nat Rev Urol 17, 513-525 (2020). 796 https://doi.org:10.1038/s41585-020-0346-4 797 67 Henao-Restrepo, A. M. et al. Efficacy and effectiveness of an rVSV-vectored vaccine in 798 preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, 799 cluster-randomised trial (Ebola Ca Suffit!). Lancet 389, 505-518 (2017). 800 https://doi.org:10.1016/S0140-6736(16)32621-6 801 68 Heppner, D. G., Jr. et al. Safety and immunogenicity of the rVSV∆G-ZEBOV-GP Ebola 802 virus vaccine candidate in healthy adults: a phase 1b randomised, multicentre, double-blind, 803 placebo-controlled, dose-response study. Lancet Infect Dis 17, 854-866 (2017). 804 https://doi.org:10.1016/S1473-3099(17)30313-4 805 69 Woolsey, C. & Geisbert, T. W. Current state of Ebola virus vaccines: A snapshot. PLoS 806 Pathog 17, e1010078 (2021). https://doi.org:10.1371/journal.ppat.1010078 807 70 Wolf, J. et al. Development of Pandemic Vaccines: ERVEBO Case Study. Vaccines (Basel) 9 808 (2021). https://doi.org:10.3390/vaccines9030190 809 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 19 71 Agnolon, V. et al. Designs and Characterization of Subunit Ebola GP Vaccine Candidates: 810 Implications for Immunogenicity. Front Immunol 11, 586595 (2020). 811 https://doi.org:10.3389/fimmu.2020.586595 812 72 Ng, T. W. & Porcelli, S. A. Designing Anti-Viral Vaccines that Harness Intrastructural Help 813 from Prior BCG Vaccination. J Cell Immunol 5, 97-102 (2023). 814 https://doi.org:10.33696/immunology.5.174 815 73 Scriba, T. J., Netea, M. G. & Ginsberg, A. M. Key recent advances in TB vaccine 816 development and understanding of protective immune responses against Mycobacterium 817 tuberculosis. Semin Immunol 50, 101431 (2020). https://doi.org:10.1016/j.smim.2020.101431 818 74 Cernuschi, T., Malvolti, S., Nickels, E. & Friede, M. Bacillus Calmette-Guerin (BCG) 819 vaccine: A global assessment of demand and supply balance. Vaccine 36, 498-506 (2018). 820 https://doi.org:10.1016/j.vaccine.2017.12.010 821 75 Gayer, M., Legros, D., Formenty, P. & Connolly, M. A. Conflict and emerging infectious 822 diseases. Emerg Infect Dis 13, 1625-1631 (2007). https://doi.org:10.3201/eid1311.061093 823 824 10 Figure Captions 825 826 Figure 1. Characterization and comparison of EBOV GP vaccines. (A) Schematic of the Th 827 GP-FL vaccine against EBOV. The Th vaccine consist of the N-terminal human Ig-kappa signal 828 sequence (hIgKss), the BCG T helper epitopes (P25 and P10) which are flanked by the cathepsin B 829 cleavage site (TVGL), GP1 which includes the mucin-like domain (MLD), the furin cleavage site, 830 GP2, and the C-terminal hexahistadine tag (6xHis). GP1 and GP2 are held together by a disulfide 831 bond. For the WT GP-FL version of the vaccine, the BCG T helper epitopes (P25 and P10) flanked 832 by the cathepsin B cleavage sites are absent. The MLD deleted versions of these vaccines were also 833 constructed (Th GPM and WT GPM). (B) SDS-PAGE under reducing conditions of purified 834 EBOV GPs as shown by Coomassie gel staining and Western blotting with anti-His antibody. The 835 GP1 and GP2 fragments, which are normally held together by disulfide bonds, were separately 836 resolved under the reducing and denaturing conditions of the SDS-PAGE analysis. The expected 837 size for GP2, which consists of the C terminal region of the EBOV GP after the MLD is 25 KDa. 838 The expected size for the GP1 precursor is 120 KDa and 60 KDa for the full length and the MLD 839 deleted version of the EBOV GP, respectively. The GP0 fragment in the full-length versions of the 840 GP constructs was detected as two or more bands of ~120-145 KDa, consistent with glycosylation 841 and disordered structure of the MLD. Purity of isolated GPs was ≥ 90% based on Coomassie blue 842 staining of the gels, and protein yields were determined by BCA protein assay (WT GP-ΔM: 2060 843 μg/ml, WT GP-FL: 220 μg/ml, Th GP-ΔM: 2972 μg/ml, Th GP-FL: 80 μg/ml). (C) ELISA with 844 antibody ADI-15878 specific for EBOV GP conformational epitope was used to probe purified 845 EBOV GPs. The ovalbumin version of the Th vaccine (Th OVA) served as a negative control to 846 show the specificity of ADI-15878 antibody against EBOV GP. (D) Processing and presentation of 847 BCG Th epitopes was shown by incubating purified EBOV GPs for 16 hours with dendritic cells and 848 in the presence of a CD4+ T cell hybridomas specific for P25 of Ag85B (left) or P10 of TB9.8 (right). 849 Supernatants were analyzed by sandwich ELISA for IL-2 (indicated as absorbance (Abs) values for 850 conversion of the assay substrate. Multiple columns were analyzed by Kruskal-Wallis one-way 851 ANOVA, followed by Dunn’s multiple comparison test; (***p < 0.001, **p < 0.01, *p < 0.05). 852 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 20 853 Figure 2. Induction of cellular and humoral immune responses by Th vaccines. For analysis of 854 cellular responses, groups of mice (n = 5) were vaccinated with BCG or received sham vaccination 855 with PBS injection, rested for 17 weeks and then immunized with the Th GP-FL or sham immunized 856 (PBS). Two weeks after the immunization, IFN ELISPOT assays were performed on unstimulated, 857 peptide-25 (P25) or Mtb (H37Rv lysate) stimulated splenocytes. (A) Representative spot forming 858 cell (SFC) images of selected animals for each group. (B) Plots showing individual animal counts 859 and group medians with interquartile range. Multiple columns were analyzed by Kruskal-Wallis one-860 way ANOVA, followed by Dunn’s multiple comparison test; (***p < 0.001 and **p < 0.01). Note 861 that values for H37Rv lysate stimulation of BCG vaccinated groups are all plotted at the upper limit 862 for accurate quantitation in the assay. (C) Mice (n = 5) were vaccinated with BCG or received sham 863 vaccination with PBS injection, rested for 5 weeks and then prime and boosted with the EBOV GP 864 vaccines (Th GP-FL or Th GPM). Two weeks after the boost, sera were collected, and antibody 865 titers against EBOV GP (WT FL) were determined using ELISA specific for IgG1 or IgG2c isotypes. 866 867 Figure 3. Expression of FcRIV increases at week 2 after BCG vaccination. (A) Flow cytometry 868 gating strategy using mice with compound genetic knock out of FcRII, RIII and RIV -chains (KO) 869 and wildtype (WT) C57BL/6 mice to determine the gating for FcRII/III and FcRIV expression on 870 immune cells. After singlet cell gating, the corresponding surface markers were used to stain 871 splenocytes to identify the following immune cells: monocytes (CD11b+ Ly6Clow), macrophages 872 (CD11b+ Ly6Chigh), neutrophils (CD11b+ Ly6G+), NK cells (NK1.1+), and B cells (B220+). (B) Mice 873 (C57BL/6) were vaccinated with 107 BCG per mouse or received PBS injections as control. Spleens 874 were harvested at week 1 after BCG vaccination (gray histogram), or at week 2 after PBS injection 875 (white histogram) or BCG (black histogram) vaccination, and splenocytes were analyzed by FACS to 876 determine the expression level of FcRIV. Top panel shows representative histograms for an 877 individual mouse from each group, and bottom panel shows median of MFI values for 5 mice in each 878 group on each indicated cell type. Median with interquartile range for five replicates is shown and 879

were analyzed using Kruskal-Wallis one way ANOVA nonparametric test and Dunn’s 880 multiple comparison test; (***p < 0.001, **p < 0.01). 881 882 Figure 4. BCG-induced FcRIV expression was maintained after Th vaccination. Mice (n = 10) 883 were sham vaccinated with PBS or with BCG at 107 CFU per mouse and rested for 17 weeks. Each 884 group was further subdivided into 2 groups (n =5) and received either PBS or the Th GP-FL vaccine 885 at 0.05 μg per mouse in alum, and splenocytes were harvested 2 weeks later to determine the level of 886 FcRIV expression by FACS. Singlet gating on splenocytes were stained for macrophages 887 (CD11bhigh Ly6Chigh), neutrophils (CD11bhigh Ly6Ghigh), and NK cells (NK1.1high) for expression of 888 FcRIV (CD16.2) as shown in (A) for representative animals of sham vaccinated with PBS and with 889 BCG alone, and quantified in (B) by MFI levels for the 5 animals in each group. The median with 890 interquartile is shown and analyzed using Kruskal-Wallis one way ANOVA nonparametric test with 891 and Dunn’s multiple comparison test; (**p < 0.01 and *p < 0.05). 892 893 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 21 Figure 5. Long lasting humoral immune response induced by the Th vaccine. (A) Serum 894 samples from sham PBS () or BCG (•) vaccinated mice and subsequently immunized once with 5 895 μg of the Th GP-FL vaccine were analyzed by ELISA for anti-EBOV GP IgG1 (left panel) or IgG2c 896 (right panel) antibodies throughout the time course of 39 weeks. Serum from a naïve mouse () 897 collected at week 2 and 4 was used to measure the background level for the ELISA assay. (B) 898 Splenocytes and bone marrow cell suspensions from these mice at 39 weeks were harvested and used 899 for B cell ELISPOT assay to quantitate anti-EBOV GP IgG1 or IgG2c antibody secreting spot 900 forming cells (SFCs). The median values with interquartile ranges are shown and analyzed using 901 Kruskal-Wallis one way ANOVA nonparametric test with Dunn’s multiple comparison test; (*p < 902 0.05). 903 904 Figure 6. BCG vaccination promotes predominantly Th1 responses. (A) Wildtype mice were 905 adoptively transferred with 4 x 104 T cells purified from P25 TCR-Tg GFP+ mice 16 hours prior to 906 vaccination with BCG (B), or peptide-25 adjuvanted with either alum (A) or LASTS-C (LC). The 907 mice were sacrificed 6 days after vaccination, and splenocytes (n=5) were analyzed by FACS for 908 master transcription regulators and key markers for Th1 (Tbet) and Tfh (CXCR-5 and Bcl-6). 909 Multiple comparisons were analyzed by Kruskal-Wallis one-way ANOVA (**p < 0.01, ***p < 910 0.001). (B) Wildtype mice were adoptively transferred with 4 x 104 T cells purified from P25 TCR-911 Tg GFP+ mice 16 hours prior to vaccination with BCG or with the P25 peptide adjuvanted with 912 LASTS-C. Formalin-fixed and paraffin-embedded spleens were cut into thin sections for 913 immunocytochemistry with anti-GFP followed by hematoxylin and eosin counterstaining. (C) High 914 magnification of boxed areas in (B) to visualize the P25 specific T cells as dark colored spots. 915 Follicles (F), germinal centers (GC), and P25 Th cells were quantified manually by a blinded 916 observer as the number of GC per follicle in (B), and the number of P25 Th cells per GC in (C). 917 Medians with interquartile ranges for 6-7 sections derived from 3 mice from each group are plotted. 918 Mann-Whitney test was used for pairwise comparison (**p < 0.01, ***p < 0.001). 919 920 Figure 7. Vaccine induced protection against lethal EBOV infection. (A) Timeline of the EBOV 921 challenge experiment. C57BL/6NHsd mice (n = 15) were vaccinated with BCG, and subsequently 922 primed and boosted with 0.05 μg of the indicated recombinant protein vaccine (WT GP-FL or the Th 923 GP-FL) in alum as indicated in the schematic. MA-EBOV infection (i.p.) with 100 pfu per mouse 924 was performed on day 98, which corresponds to day 0 (blue) of the challenge experiment. Arrows 925 represent blood sample collections, and harvesting of blood, livers, and spleens at the last collection 926 point after sacrifice. (B) Serum samples collected 2 weeks after administering WT GP-FL (WT) or 927 the Th GP-FL (Th) vaccines for both priming and boosting time-points were analyzed by ELISA for 928 anti-EBOV GP IgG1 (red) and IgG2c (blue) antibodies. (C) Endpoint titers of anti-EBOV GP 929 antibodies following boosting with the WT or Th vaccines were also analyzed by ELISA in sera from 930 mice primed and boosted with either PBS or 0.05 μg of the indicated recombinant protein vaccine. 931 (D) Naïve mice (black) or mice that received the WT (red) or Th (blue) vaccines were challenged 932 with MA-EBOV and the times to death (left panel) and body weight changes (right panel) for 933 individual mice were recorded. The Mantel-Cox test was performed for comparison of the survival 934 curves; (****p < 0.0001). (E) Five days after the challenge, mice (n = 5) were sacrificed to 935 determine viral titers from the corresponding organs. The median with interquartile ranges is shown 936 and analyzed using Kruskal-Wallis one way ANOVA nonparametric test with and Dunn’s multiple 937 comparison test; (**p < 0.01, *p < 0.05). (F) The anti-EBOV GP IgG1 and IgG2c antibody titers 938 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint 22 were also measured from serum of these mice sacrificed at day 5 after challenge (****p < 0.0001, 939 **p < 0.01, *p < 0.05 two-way ANOVA test and Sidak’s multiple comparison test). (G) Twenty-940 eight days after the challenge, the surviving mice were sacrificed, and blood was collected to measure 941 anti-EBOV GP IgG1 and IgG2c antibody titers. 942 943 11 Supplementary Material Captions 944 945 Supplemental Figure 1. Analysis of EBOV GP by size exclusion chromatography. Purified 946 wildtype EBOV GP full-length (WT GP-FL) and the Th EBOV GP vaccine (Th GP-FL) were 947 subjected to the SRT SEC-300 size exclusion column. SRT SEC-300 exclusion limit is 1250 kDa. 948 949 Supplementary Figure 2. Comparison of EBOV GP vaccines. (A) ELISA with antibody KZ52 950 specific for EBOV GP conformational epitopes was used to probe purified EBOV GP. The 951 ovalbumin version of the Th vaccine (Th OVA) served as a negative control to show the specificity 952 of KZ52 antibody against EBOV GP (left panel). 953 954 Supplemental Figure 3. Susceptibility of 24-week old C57BL/6N to MA-EBOV challenge. 24-955 week old mice were infected with 10 or 1000 FFU of mouse-adapted (MA-) EBOV and succumbed 956 to infection by day 7-8 after EBOV challenge. 957 958 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 31, 2024. ; https://doi.org/10.1101/2024.05.28.595735doi: bioRxiv preprint