key: cord-1012772-qi4ovke4 authors: Coria, Lorena M.; Saposnik, Lucas M.; Castro, Celeste Pueblas; Castro, Eliana F.; Bruno, Laura A.; Stone, William B.; Pérez, Paula S.; Darriba, M. Laura; Chemes, Lucia B.; Alcain, Julieta; Mazzitelli, Ignacio; Varese, Augusto; Salvatori, Melina; Auguste, Albert J.; Álvarez, Diego E; Pasquevich, Karina A.; Cassataro, Juliana title: A novel bacterial protease inhibitor adjuvant in RBD-based COVID-19 vaccine formulations increases neutralizing antibodies, specific germinal center B cells and confers protection against SARS-CoV-2 infection date: 2021-12-09 journal: bioRxiv DOI: 10.1101/2021.12.07.471590 sha: 426036fedd434e1019dd331446f92bfbc20eb36f doc_id: 1012772 cord_uid: qi4ovke4 In this work we evaluated recombinant receptor binding domain (RBD) based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus Alum, AddaS03, AddaVax or the combination of Alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and Alum as adjuvants have a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus Alum, AddaVax or AddaS03. Antibodies induced with the formulation containing U-Omp19 not only increased their neutralization capacity against the wild-type virus but also cross neutralized alpha, lambda and gamma variants with similar potency. Also, addition of U-Omp19 to vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+Alum formulation induced RBD-specific Th1 and CD8+ T cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal SARS-CoV-2 challenge of K18-hACE2 mice. ). This increment was statistically significant (p=0.0257 vs RBD+Alum, p =0.0259 vs 167 AddaVax and p =0.022 vs AddaS03). AddaVax or AddaS03 as adjuvants induced a titer of 168 neutralizing antibodies similar to Alum alone (Fig. 3B) . 169 To assess the functionality of vaccine-elicited antibodies against the wild-type SARS-CoV-2, 170 neutralization assay with sera from immunized animals was performed. Similar to the results 171 obtained using the pseudovirus system, one month after the second dose, RBD+Alum+U-Omp19 172 immunized mice had significant higher virus neutralization antibody titers in serum (Fig. 3C , GMT 173 612.1 95%CI 87.80-4267) than mice immunized with RBD+Alum (Fig. 3C , GMT 140 95%CI 16.34-174 1199) or plus commercial adjuvants (AddaVax or AddaS03). These results further confirm the data 175 obtained by PsVNA. 176 As SARS-CoV-2 initially infects the upper respiratory tract, its first interactions with the 177 immune system must occur predominantly at the respiratory mucosal surfaces. Mucosal responses 178 may be crucial to stop person to person transmission of this virus. Thus, examination of neutralizing 179 activity in BAL was performed using pseudotyped virus system. Of note, adding U-Omp19 as an 180 adjuvant to the formulation increased wild-type virus neutralization in the BAL of mice compared 181 with the vaccine adjuvanted with Alum alone, AddaVax or AddaS03 (Fig. 3D , *p<0.05). 182 Duration of vaccine immunity is key to estimate how long protection lasts. To this effect, we 184 evaluated the level of total antibodies over 175 days after prime immunization with the vaccine 185 formulation containing U-Omp19 and alum as adjuvants. Interestingly, titers of anti-RBD IgG 186 antibodies remained stable at least 5-6 months after i.m. immunization of mice with this formulation 187 ( Fig. 4A) . Remarkably, neutralizing capacity of the antibodies remained stable till day 175 post 188 prime immunization (Fig. 4B ) 189 variants To adequately address the public health impact that newly emerging present, there is a need for vaccine-elicited antibodies that can cross-neutralize different CoV-2 variants. Thus, neutralization activity of sera against prevalent circulating variants of CoV-2 in our region: alpha (B.1.1.7, first identified in UK), gamma (P.1, first identified in Manaos, 195 Brazil) and lambda (C.37, first identified in Peru) was evaluated and compared to neutralizing 196 activity against wild-type reference strain. In particular, gamma and lambda variants have been 197 shown to partially escape neutralization by antibodies triggered by previously circulating variants or 198 vaccine induced antibodies 25, 26 . Noteworthy, antibodies induced after vaccination with the 199 formulation containing U-Omp19 not only neutralize the wild-type virus but also cross neutralized 200 alpha, lambda and gamma variants (Fig. 5) . In contrast, antibodies produced by mice immunized 201 with RBD+Alum could neutralize the wild-type SARS-CoV-2 and alpha variant but showed 202 significantly lower neutralizing activity against gamma and lambda variants (Fig. 5) . AddaVax and 203 AddaS03 adjuvanted formulations induced similar neutralizing antibody titers against the wild-type, 204 gamma and lambda variants (Fig. 5) . 205 Altogether these results demonstrate that addition of U-Omp19 to the Alum plus RBD 206 vaccine formulation increases virus neutralizing antibodies, specific IgA in BAL and neutralizing 207 antibodies of the virus in BAL. Neutralizing antibodies are proposed as the best correlate of 208 protection thus we focused the next studies on the vaccine formulation containing U-Omp19 as 209 adjuvant. 210 lung 212 In addition to memory B cells and neutralizing antibodies, induction of specific T cell immune 213 responses could have a role in protection against SARS-CoV-2 infection 27 . 214 To determine T cell-mediated immune responses, splenocytes and lung cells from 215 RBD+Alum or RBD+Alum+U-Omp19 immunized mice were stimulated with RBD or medium alone 216 and then cytokines levels in the supernatants were measured. Both formulations were able to 217 induce Ag-specific cytokine secretion at spleen (Fig. 6A) . Importantly, the levels of interferon (IFN)-y 218 were higher than interleukin (IL)-5 at spleens of both vaccine formulations. In lungs, immunization 219 with RBD+Alum+U-Omp19 promoted a significant increment in IFN-y secretion compared with the 220 formulation containing RBD+Alum (Fig. 6B) . However, the Alum-adjuvanted vaccine elicited a 221 higher amount of IL-5 by lung (Fig. 6B) . These results suggest that U-Omp19 as adjuvant promotes 222 a specific T cell response biased to a Th1 profile in the lung. 223 To further evaluate the Th1/2 balance, IFN-γ and IL-4 producing cells were measured by 224 intracellular cytokine staining. Spleen cells from immunized mice were stimulated with a pool of 225 SARS-CoV-2 RBD peptides to detect antigen-specific T cell responses. Percentages of IFN-γ-226 producing CD4 + and CD8 + T cells were increased in both groups of mice while IL-4-producing CD4 + 227 T cells were only increased after RBD+Alum administration ( Fig. 6C and D) . These results support 228 the induction of Th1 and CD8 + T cell immune responses after immunization with RBD adjuvanted 229 with U-Omp19 in combination with Alum. 230 Immunogenicity of vaccine formulations using Alum alone or combining both adjuvants 232 (alum and U-Omp19) with RBD as Ag was also evaluated in the C57BL/6 mouse strain. Vaccine 233 formulations were administered following the same schedule used for BALB/c mice, two doses 234 every 14 days. 235 Both vaccine formulations induced high anti-RBD IgG titers in sera (Fig. 7A) . Remarkably, 236 anti-RBD IgA levels in the BAL of RBD+Alum+U-Omp19 immunized mice were higher than in the 237 RBD+Alum immunized mice (Fig. 7B , *p<0.05). There were no differences in specific IgG levels in 238 the BAL between both groups (Fig. 7B) . 239 Formulation containing RBD+U-Omp19+Alum induced higher neutralizing antibody titers 240 than RBD+Alum formulation (Fig. 7C Addition of U-Omp19 to RBD plus Alum vaccine increased the neutralizing antibody titers 243 against authentic wild-type virus. Four weeks after second dose, sera of mice immunized with RBD+Alum+U-Omp19 produced a ten-fold increase in the viral neutralizing antibodies titer (GMT 245 929 95%CI 37.50-10079 Fig. 7D ) compared with mice receiving RBD + Alum (Fig. 7D , GMT 99 246 95%CI 13.61-210.1). These results validated the data obtained in BALB/c mice and further indicate 247 that U-Omp19 can be used under different genetic backgrounds. 248 plasmablasts in the spleen 250 A persistent germinal center (GC) B cell response enables the generation of robust humoral 251 immunity 28 . Therefore, specific GC B cells were evaluated in spleens from vaccinated mice one 252 month after second dose, Fig. 8A shows the gating strategy used. There were no differences 253 between groups in the total CG cells (B220 + CD19 + IgD -CD95 + GL7 + cells) among spleen samples 254 ( Fig. 8B) . Of note, there were differences in the frequency of RBD + specific GC cells as mice 255 immunized with RBD+Alum+U-Omp19 increased the percentage of RBD + specific GC cells in 256 comparison with RBD+Alum (Fig. 8C) . Besides, the percentages of RBD + specific plasma blasts 257 (B220 + CD19 + IgD -CD138 + cells) were also higher in animals from RBD+Alum+U-Omp19 than from 258 RBD+Alum (Fig. 8D) . These results indicate a better performance of the vaccine formulation 259 containing U-Omp19 to induce specific GC and secretory B cells one month after immunization. 260 To determine vaccine efficacy, we used a severe disease model using K-18-hACE2 262 transgenic mice. Infection of transgenic mice with SARS-CoV-2 results in lung disease with signs of 263 diffuse alveolar damage, and variable spread to the central nervous system 29 . The lethal dose 50% 264 (LD50), is estimated to be 10 4 plaque-forming units (PFU) 30 . Vaccine formulation efficacy was 265 evaluated in K18-hACE2 mice vaccinated with RBD+Alum+U-Omp19 or PBS (control) and 266 challenged intranasally with 2x10 5 PFU of SARS-CoV-2. At day 5 post infection some animals were 267 euthanized to assess the viral load in lungs and brains. The presence of the SARS-CoV-2 virus was 268 not detected in the lungs while very low virus titers were detected in the brains of animals 269 vaccinated with RBD+Alum+U-Omp19 (Fig. 9A) . In contrast a high viral load was detected in the 270 lungs and brains of animals immunized with PBS (Fig. 9A) . It is noteworthy that the majority of the 271 mice vaccinated with RBD+Alum+U-Omp19 did not lose weight after challenge (Fig. 9B) not been reported for the existing COVID-19 vaccines, a recent study has shown that antibodies 296 against the S protein N-terminal domain enhanced the binding capacity of S protein to ACE2 and 297 infectivity of SARS-CoV-2 38 . To mitigate the ADE effect, minimizing non-neutralizing epitopes we 298 decided to work with the SARS-CoV-2 spike protein RBD. Furthermore, in our hands (data not 299 shown) and as described by others 39 , yields of recombinant RBD were much higher than those of 300 full-length Spike, an important factor in delivering the vaccine to global population. The protocols performed were also approved by the Institutional Committee for the use and care of 450 experimental animals (CICUAE) from National University of San Martin (UNSAM) (01/2020). 451 Eight-week-old female BALB/c or C57BL/6 mice were obtained from IIB UNSAM animal facility. was observed in the well, this well was considered as a well with a manifestation of CPE. 522 Neutralization titer was defined as the highest serum dilution without any CPE in two of three 523 replicable wells. Otherwise, plates were scanned for determination of media absorbance at 585 nm 524 and non-linear curves were fitted to obtain the titer corresponding to the 50% of neutralization 525 (NT50). Neutralization assays to compare neutralization among different SARS-CoV-2 variants 526 (alpha, gamma and lambda) were performed in the same plate for each sample. 527 Four weeks after the second dose, mice were sacrificed to study cellular responses. Intracellular 529 cytokine determination: splenocytes were cultured (4x10 6 cells/well) in the presence of stimulus 530 medium (complete medium supplemented with anti-CD28 and anti-CD49d) or Ag stimuli (stimulus 531 medium + RBD-peptides + RBD protein) for 18 h. Next, brefeldin A was added for 5 h to the 532 samples. After that, cells were washed, fixed, permeabilized, stained, and analyzed by flow Software, San Diego, CA). In experiments with more than two groups, data were analyzed using 555 one-way ANOVA with a Bonferroni post-test. When necessary, a logarithmic transformation was 556 applied prior to the analysis to obtain data with a normal distribution. In experiments with two 557 groups, an unpaired t test or Mann-Whitney U test were used. A p value <0.05 was considered 558 significant. When bars were plotted, results were expressed as means ± SEM for each group. 559 The authors declare that the data supporting the findings of this study are available from the 561 corresponding author upon reasonable request. 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Characterization of recombinant RBD. A. Coomassie blue-SDS-PAGE stained of reduced RBD Western Blot analysis of recombinant RBD produced in HEK-293 cells using a convalescent 765 human serum as primary antibody. M: page ruler. C. Representative size exclusion chromatography elution 766 profile of recombinant RBD. Black bars represent MWM Aprotinin (6.5 KDa), Vo and Vo+Vi are shown as dashed lines Binding of recombinant RBD (rRBD) to HEK-293T cells expressing hACE2. HEK-293T were used as control Histograms and percentage of cells positive for RBD are shown Figure 2. BALB/c mice immunized with RBD plus different adjuvants induced RBD-specific antibodies Serum samples were 787 obtained at indicated time points for ELISA and neutralization assays. B. Kinetics of RBD-specific IgG 788 endpoint titers in sera of immunized animals by ELISA. Points are means ± SEM. C. RBD-specific IgG 789 subclasses (IgG1 and IgG2a) titers in sera Detection of RBD-specific IgG (D) and IgA (E) in the bronchoalveolar lavage of immunized mice at day 42 791 post prime immunization neutralization (NT50). B. Kinetics of neutralizing antibody titers of all groups SARS-CoV-2 assay. Points are Means ± SEM. Titers correspond to the 50% of virus neutralization (NT50) One way ANOVA with Bonferroni post-test. C. Neutralizing antibody titers against wild-type SARS-CoV-2 virus at day 42 post prime immunization. Neutralization titer was defined as the highest serum 814 dilution without any cytopathic effect in replicable wells (NT 90). Data are shown as means ± SEM One way ANOVA with Bonferroni post-test. D. Determination of neutralizing antibodies in the 816 bronchoalveolar lavage by pseudo-typed SARS-CoV-2 assay at day 42. Data are expressed in percentage of 817 neutralization compared with controls (virus alone) 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863