key: cord-0787183-5zj6f9rh authors: Haun, Brien K.; Lai, Chih-Yun; Williams, Caitlin A.; Wong, Teri Ann; Lieberman, Michael M.; Pessaint, Laurent; Andersen-Elyard, Hanne; Lehrer, Axel T. title: CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation date: 2020-07-26 journal: bioRxiv DOI: 10.1101/2020.07.24.220715 sha: 3f69b0e77b70b5b603b42d56493f051764ddabec doc_id: 787183 cord_uid: 5zj6f9rh The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT™ or Alhydrogel. CoVaccine HT™ induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralising antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT™ can rapidly induce a comprehensive and protective immune response to SARS-CoV-2. In order to investigate which adjuvants induce a strong humoral response, our group has 47 formulated protein subunit vaccine candidates using a recombinant SARS-CoV-2 Spike 48 subdomain 1 (S1) protein, obtained from Sino Biological, Inc., adjuvanted with either CoVaccine CoVaccine HT TM also offers an advantage in comparison to Alhydrogel regarding particle size. Alhydrogel particles typically fall within the range of 1-10 microns 14 whereas CoVaccine HT TM 66 is typically showing droplet sizes around 130 nm 15,16 . Smaller particle sizes offer increased 67 stability and enhanced adjuvanticity and in comparison, particle sizes of other commercial stable 68 oil-in-water emulsion adjuvants (MF59 and AS03) are in the range of 160nm 17 . These oil-in-69 water emulsion adjuvants utilize squalene, a shark fat derived product 9,18,19 . The use of squalane 70 in CoVaccine HT TM as a plant-derived product may be advantageous due to availability, reduced 71 regulatory burden, and potentially also ideologically to the population being immunised. In 72 summary, CoVaccine HT TM could provide a distinct advantage over Alhydrogel as the more 73 conventional adjuvant choice. Here we tested the immunogenicity of SARS-CoV-2 Spike S1 proteins adjuvanted with either 76 CoVaccine HT™, Alhydrogel, or phosphate buffered saline (PBS) in BALB/c mice. We assessed 77 overall antibody titres, immunoglobulin subclass diversity, cell mediated immunity, and in-vitro 78 neutralisation of wild-type SARS-CoV-2 virus. We demonstrate that CoVaccine HT™ elicits 79 rapid humoral responses, increased subclass diversity, more interferon gamma (INFγ) 80 production, and higher neutralising antibody titres than the other adjuvants. Collectively, CoVaccine HT™ may be advantageous over other adjuvants for a SARS-CoV-2 vaccine. An additional serum sample was collected by cardiac puncture at day 28 along with splenocytes 92 from three animals in the Spike S1 + CoVaccine HT™ (S1+CoVac) and S1 + Alum groups, and 93 two animals in the S1+PBS group. A PRNT was performed in a biosafety level 3 facility (at BIOQUAL, Inc.) using 24-well plates. Mouse sera pooled from individual mice within each group, were diluted to 1:10, and a 1:3 serial Murine immunisation with SARS-CoV-2 Spike S1 proteins 163 Neutralising antibodies of SARS-CoV-2 largely target the receptor binding domain present 164 within the Spike S1 protein 24 . Therefore, BALB/c mice were given two doses of commercially 165 available Spike S1, 21 days apart (Fig.1A) . To test whether adjuvants may alter immunological 166 responses to the immunogen, mice were divided into four groups based on vaccine formulation. The mice receiving S1 protein and CoVaccine HT™ (S1+CoVac), Alhydrogel (S1+Alum), or 168 PBS (S1+PBS) received SARS-CoV-2 Spike S1 mixed with either CoVaccine HT™ ("CoVac"), 169 Alhydrogel ("Alum"), or PBS, respectively. One group received CoVaccine HT™ alone as an 170 adjuvant control (Fig. 1A) . Adjuvants alter immunogenicity and specificity to immunisation 173 Serum analysis revealed high reactivity of SARS-CoV-2 S1 specific IgG antibodies in 174 S1+CoVac after a single dose while S1+Alum titres were near baseline (Fig.1B) . Only one 175 animal showed a detectable titre in the antigen alone group at this time point. Only in the group 176 with CoVaccine HT™ a low level of cross reactivity was observed after the first dose to SARS-177 CoV S1. On day 35, S1+Alum and S1+PBS displayed significantly higher antibody responses 178 compared to day 14 and variations among individual animals were reduced. S1+CoVac treated 179 animals on day 35 consistently showed very high antibody responses in every animal. Similarly, 180 cross-reactivity with SARS-CoV S1 was greatly increased for all groups on day 35 (Fig.1B) . As 181 expected, due to its much lower sequence homology, the SARS-CoV-2 S1 did not induce IgG 182 responses to MERS-CoV S1. In patients suffering from COVID-19, high RBD-specific IgG titres have been observed 25 . However, higher titres of SARS-CoV-2 Spike-specific IgG are associated with patients that did 186 not require intensive care unit treatment while lower titres are associated with increased disease 187 severity 26 . Therefore, the antibody response kinetics may be an important factor for a successful 188 vaccine candidate. Time-course analysis of IgG responses reveal that adjuvanted S1 may be 189 crucial for strong, early IgG responses with SARS-CoV-2 specificity while a second dose may 190 decrease variability among individual animals and increase cross-reactivity (Fig.1C) . CoVaccine HT™ improves IgG titres to SARS-CoV-2 and SARS-CoV S1 proteins 193 To further investigate the matured IgG responses, sera from day 35 were titrated in a four-fold 194 dilution series starting at 1/250 and analysed by microsphere immunoassay (MIA). The 195 S1+Alum and S1+PBS groups showed reactivity to SARS-CoV-2 S1 when diluted up to 196 1/256,000, indicating an abundance of antigen-specific IgG in the sera ( Fig. 2A) . Titrating sera 197 from S1+CoVac however, revealed saturating levels of IgG for five dilutions and detectable IgG 198 levels were present down to a 1/65.5 million dilution. Antiserum to S1+CoVac also showed 199 significantly greater cross reactivity to SARS-CoV S1 compared to the other groups. All groups 200 remained negative for cross reactivity to MERS-CoV S1 (Fig.2A) . These data suggest that 201 immunisation with SARS-CoV-2 S1 and CoVaccine HT™ elicits robust antigen-specific IgG 202 response with the expected cross-reactivity profile to include SARS-CoV S1. Th1 or Th2 response may have been more prominent. Therefore, sera from each S1+adjuvant 209 group were analysed for their subclass composition (Figure 3) . Consistent with previous 210 findings, the S1+CoVac group displayed a diverse immunoglobulin response composed of IgG1, 211 IgG2a, and IgG2b subclasses all of which were further elevated after a second dose of vaccine. Low levels of IgG3 were also observed. Alternatively, the Alum and antigen alone groups 213 primarily produced an IgG1 response with some detectable IgM in the Alum group, representing 214 a classical Th2-biased humoral response. Heterogeneous subclass populations such as those 215 observed in the S1+CoVac group are typically associated with Th1 responses while IgG1 is 216 characteristic of a Th2 response. To further investigate the nature of these adjuvant effects, the 217 subclass data were stratified to analyse ratios of Th1 vs Th2 subclasses ( Figure 3C ). This analysis 218 clearly shows that of the three tested formulations, only S1+CoVac induced a relatively balanced 219 humoral response. Furthermore, only the S1+CoVac formulation was able to induce detectable 220 SARS-CoV-2 neutralising antibody titres as demonstrated in a plaque reduction neutralisation 221 test using wildtype virus (Table 1 ). PRNT90 and PRNT50 titres for this formulation indicate 222 potent neutralisation (1:1620). Adjuvant effect on the SARS-CoV-2 S1-specific INFγ responses 225 We assessed the adjuvant effect of CoVaccine HT™ and Alum on the cellular immune responses 226 directed against SARS-CoV-2 S1 using an IFN- FluoroSpot assay. Individual mouse spleens 227 from each group harvested at day 7 post-second immunisation were processed, and single cell 228 suspensions stimulated with SARS-CoV-2 S1 peptides. The number of IFN- secreting cells 229 from the mice given CoVaccine HT™ was significantly higher than those for mice given Alum 230 or S1 antigen only at two different peptide concentrations (Figure 4) . Splenocytes from 231 unvaccinated (naïve) mice did not respond to S1 peptide stimulation with only 2 spot forming 232 cells (SFCs)/10 6 cells detected. The results suggest that CoVaccine HT TM is a superior adjuvant 233 for induction of an antigen-specific Th1-focused cellular immune response, which is critical for 234 SARS-CoV-2 vaccine development. The high potency for SARS-CoV-2 S1 in the CoVaccine HT TM formulation may be attributable 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 Figure 1 . Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study 366 design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to 367 custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-421 CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 ( 422 one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were 423 used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 424 peptide pools at 0.2 g/mL or 0.5 g/mL per peptide or medium. IFN- secreting cells were 425 enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the 426 number of spot forming cells (SFC)/10 6 splenocytes after subtraction of the number of spots 427 formed by cells in medium only wells to correct for background activity. *** p  0.001, **** p  428 0.0001. 429 430 431 Table 1 . SARS-CoV-2 neutralisation titres 432 433 Group ID Titre (PRNT 90 ) Titre (PRNT 50 ) S1+CoVac 1620 1620 S1+Alum <20 <20 S1+PBS <20 <20 CoVaccine HT <20 <20 434 435 World Health Organization. Coronavirus disease (COVID-19) Situation Unique epidemiological and clinical features of 463 the emerging 2019 novel coronavirus pneumonia (COVID-19) implicate special control 464 measures SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is 466 Blocked by a Clinically Proven Protease Inhibitor Characterization of the receptor-binding domain (RBD) of 2019 novel 469 coronavirus: implication for development of RBD protein as a viral attachment inhibitor 470 and vaccine Neutralizing antibody and 472 protective immunity to SARS coronavirus infection of mice induced by a soluble 473 recombinant polypeptide containing an N-terminal segment of the spike glycoprotein The COVID-19 vaccine development landscape COVID-19 TREATMENT AND VACCINE TRACKER An empirical approach towards the efficient and optimal production 480 of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel 481 adjuvant CoVaccine HT is functionally superior to Freund's adjuvant Sucrose fatty acid sulphate esters as novel vaccine 484 adjuvants: effect of the chemical composition Alhydrogel(R) adjuvant, ultrasonic dispersion and protein binding: a 487 TEM and analytical study Analysis of the role of vaccine adjuvants in 489 modulating dendritic cell activation and antigen presentation in vitro Overcoming CD4 Th1 Cell Fate Restrictions to Sustain Antiviral CD8 T 492 Cells and Control Persistent Virus Infection Recombinant Zika Virus Subunits Are Immunogenic and Efficacious in Mice Reprogramming the adjuvant properties of aluminum oxyhydroxide with 497 nanoparticle technology A novel non-mineral oil-based adjuvant. I. Efficacy of a 500 synthetic sulfolipopolysaccharide in a squalane-in-water emulsion in laboratory animals Sucrose fatty acid sulphate esters as novel vaccine 503 adjuvant The droplet size of emulsion adjuvants has significant impact on their 505 potency, due to differences in immune cell-recruitment and -activation Squalene and squalane emulsions as adjuvants Development and evaluation of AS03, 510 an Adjuvant System containing alpha-tocopherol and squalene in an oil-in-water 511 emulsion Effect of serum heat-513 inactivation and dilution on detection of anti-WNV antibodies in mice by West Nile virus 514 E-protein microsphere immunoassay Detection of human anti-flavivirus antibodies with a west nile virus 517 recombinant antigen microsphere immunoassay Serological evidence of Ebola virus exposure in dogs from affected 519 communities in Liberia: A preliminary report Infection with non-lethal West 522 Nile virus Eg101 strain induces immunity that protects mice against the lethal West Nile 523 virus NY99 strain Neutralizing Antibodies against SARS-CoV-2 and Other 525 Human Coronaviruses Serum IgA, IgM, and IgG responses in COVID-19 Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 530 patients Requirement of TLR4 signaling for the induction of a Th1 immune 533 response elicited by oligomannose-coated liposomes Th1 disabled function in response to TLR4 stimulation of 536 monocyte-derived DC from patients chronically-infected by hepatitis C virus Cutting edge: inflammasome activation by 539 alum and alum's adjuvant effect are mediated by NLRP3 Role of aluminum-containing adjuvants in antigen internalization by 542 dendritic cells in vitro What is an adjuvant and why is it added to a vaccine? Potential adjuvants for the development of a SARS-CoV-2 547 vaccine based on experimental results from similar coronaviruses Evaluation of Antibody-Dependent Enhancement of SARS-CoV Infection in 550 Rhesus Macaques Immunized with an Inactivated SARS-CoV Vaccine Immunization with SARS coronavirus vaccines leads to pulmonary 553 immunopathology on challenge with the SARS virus A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in 556 Recombinant proteins of Zaire ebolavirus induce potent humoral and 558 cellular immune responses and protect against live virus infection in mice Vaccination with Plasmodium knowlesi AMA1 formulated 561 in the novel adjuvant co-vaccine HT protects against blood-stage challenge in rhesus 562 macaques Safety and immunogenicity of multi-antigen AMA1-based vaccines 564 formulated with CoVaccine HT and Montanide ISA 51 in rhesus macaques Convergent antibody responses to SARS-CoV-2 in convalescent 567 individuals Antibody signature induced by SARS-CoV-2 spike protein 569 immunogens in rabbits Comparison of multiple adjuvants on the stability and immunogenicity 571 of a clade C HIV-1 gp140 trimer The potential danger of suboptimal antibody responses in 574 COVID-19 Anti-severe acute respiratory syndrome coronavirus spike antibodies 576 trigger infection of human immune cells via a pH-and cysteine protease-independent 577 FcgammaR pathway Antibody-dependent enhancement of SARS coronavirus infection and its 579 role in the pathogenesis of SARS Priming immunization determines T helper cytokine mRNA 581 expression patterns in lungs of mice challenged with respiratory syncytial virus Respiratory syncytial virus disease in infants despite prior 584 administration of antigenic inactivated vaccine T cell responses to whole SARS coronavirus in humans Memory T cell responses targeting the SARS coronavirus persist up to 590 11 years post-infection Cellular immune responses to severe acute respiratory syndrome 593 coronavirus (SARS-CoV) infection in senescent BALB/c mice: CD4+ T cells are 594 important in control of SARS-CoV infection T cell responses are required for protection from clinical 597 disease and for virus clearance in severe acute respiratory syndrome coronavirus-598 infected mice Virus-specific 600 memory CD8 T cells provide substantial protection from lethal severe acute respiratory 601 syndrome coronavirus infection Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans 604 with COVID-19 Disease and Unexposed Individuals 436 The authors have no known conflicts of interest.