CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation

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.

65
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.

81
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.

127
A PRNT was performed in a biosafety level 3 facility (at BIOQUAL, Inc.) using 24-well plates.

128
Mouse sera pooled from individual mice within each group, were diluted to 1:10, and a 1:3 serial

162
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.

172
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 .

185
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).

192
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.

212
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).

224
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.