key: cord-0919958-e1qf75mh
authors: Rice, Adrian; Verma, Mohit; Voigt, Emily; Battisti, Peter; Beaver, Sam; Reed, Sierra; Dinkins, Kyle; Mody, Shivani; Zakin, Lise; Sieling, Peter; Tanaka, Shiho; Morimoto, Brett; Higashide, Wendy; Olson, C. Anders; Gabitzsch, Elizabeth; Safrit, Jeffrey T.; Spilman, Patricia; Casper, Corey; Soon-Shiong, Patrick
title: Heterologous Vaccination with SARS-CoV-2 Spike saRNA Prime followed by DNA Dual-Antigen Boost Induces Robust Antibody and T-Cell Immunogenicity against both Wild Type and Delta Spike as well as Nucleocapsid Antigens
date: 2021-11-30
journal: bioRxiv
DOI: 10.1101/2021.11.29.470440
sha: 93a9afa4d70719d2431ac90a7ce0f9580b9c4450
doc_id: 919958
cord_uid: e1qf75mh

We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment to increase the potential for MHC class I and II stimulation. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by prime > boost homologous and heterologous combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG against wild type S1 and Delta (B.1.617.2) variant S1 was generated at similar levels. An AdS+N boost of an SASA S prime enhanced both CD4+ and CD8+ T-cell responses to both S wild type and S Delta peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus tested: Wuhan, Delta, and Beta strain pseudoviruses. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against COVID-19 and SARS-CoV-2 variants.

Impressive efforts of the scientific and pharmaceutical community have resulted in the design, 48 testing and successful deployment of several COVID-19 vaccines that have shown high levels of 49 efficacy. 1-5 Nonetheless, SARS-CoV-2 viral variants have continued to emerge and spread 50 throughout the globe, particularly in areas where vaccination rates are low or vaccines are 51 unavailable. 52

To address the need for a vaccine regimen that would be highly efficacious against 53

predominating and emerging variants that may be made available in currently underserved areas 54 and nations, and leverages the resilience of cell-mediated immunity against variants, we previously 55 developed a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) 56 plus nucleocapsid (N) vaccine (AdS+N). 6, 7 This vaccine encoding Wuhan strain or 'wild type' 57 (wt) SARS-CoV-2 S modified with a fusion sequence (S-Fusion) to enhance cell-surface 58 expression 6,7 as well as N modified with an Enhanced T-cell Stimulation Domain (N-ETSD) 8 to 59 increase the potential for MHC class I and II stimulation 9-11 has been shown to elicit humoral and 60 T-cell responses in mice, 7 non-human primates (NHP), 6 and participants in Phase 1b trials. 8 The 61

Ad5S+N vaccine given as a subcutaneous (SC) prime with two oral boosts protected NHP from 62 SARS-CoV-2 infection 6 and a single prime vaccination of clinical trial participants generated T-63 cell responses that were sustained against a series of variant S peptide sequences, including those 64 for the B.1.351, B.1.1.7, P.1, and B.1.426 variants. 8 65

Despite the promising findings with the AdS+N vaccine candidate, we wish to continue to 66 investigate vaccine regimens with the potential to maximize immune responses -both humoral 67 and cellular. One such approach is by heterologous vaccination utilizing two nucleic acid-based 68 vaccines: ImmunityBio's hAd5 vectored DNA vaccine and the Infectious Disease Research 69

Institute's (IDRI's) RNA-based vaccine. 12 Heterologous vaccination using vaccine constructs 70 expressing the same or different antigens vectored by different platforms, specifically 71 combinations of RNA-and adenovirus-based vaccines has previously been reported to 72 significantly increase immune responses. 13,14 73 To assess the potential for enhanced immune responses by heterologous vaccination, we tested 74 prime > boost combinations of the AdS+N vaccine with a self-amplifying and self-adjuvanted 75 S(wt) RNA-based vaccine (SASA S) delivered in a nanostructured lipid carrier (NLC) . 15, 16 The 76 NLC stabilizes the self-amplifying RNA [17] [18] [19] and delivers it to cells wherein it is amplified and the 77 S protein expressed. The S sequence in the SASA S vaccine comprises a codon-optimized 78 sequence with the D614G mutation 20 that increases SARS-CoV-2 susceptibility to neutralization, 79 21 a diproline modification to stabilize S in the pre-fusion conformation that increases antigenicity, In this work, the two aforementioned vaccines were tested by homologous prime > boost 85 delivery of each as compared to heterologous delivery regimens with an alternating order: AdS+N 86 > SASA S and SASA S > AdS+N. The findings reported here support our hypothesis that 87 heterologous vaccination with the SASA S and AdS+N vaccines would enhance immune 88 responses, particularly T-cell responses. 89

Both CD4+ and CD8+ T-cell responses were enhanced by heterologous vaccination, with 90 CD4+ interferon-g (IFN-g) production in response to both S(wt) peptides being higher with the 91 SASA S prime > AdS+N boost combination as compared to all other groups. Notably, CD4+ and 92 CD8+ T cells were equally responsive to S(wt) and S(Delta) peptides and responses of T cells 93 from SASA S > AdS+N to S(Delta) were also the highest of the groups. 94

Findings were similar for unselected T cells in ELISpot analyses, which again revealed the 95 SASA S > AdS+N combination resulted in significantly higher IFN-g secretion by T cells in 96 response to both S(wt) peptides than all other groups. 97

We further demonstrate that all combinations that included the SASA S vaccine elicited the 98 greatest anti-full length (FL) S wild type (wt), anti-S1(wt) and -importantly-anti-Delta variant 99 (B.1.617.2) S1 IgG responses. Regimens comprising the SASA S vaccine also generated sera that 100 showed high and similar capability to neutralize Wuhan, Delta, and Beta strain pseudovirus. 101

As expected, anti-N IgG antibodies and T-cell responses to N peptides were seen only for 102 vaccine combinations that delivered the N antigen and were very similar among groups receiving 103 the AdS+N vaccine in any order. 104

The hAd5 [E1-, E2b-, E3-] The SASA S vaccine comprises an saRNA replicon composed of an 11.7 kb construct 117 expressing the SARS-CoV-2 Spike protein, along with the non-structural proteins 1-4 derived from 118 the Venezuelan equine encephalitis virus (VEEV) vaccine strain TC-83. The Spike RNA sequence 119 is codon-optimized and expresses a protein with the native sequence of the original Wuhan strain 120 plus the dominant D614G mutation, with the prefusion conformation-stabilizing diproline (pp) 121 mutation (consistent with other vaccine antigens) and replacement of the furin cleavage site RRAR 122 sequence with a QQAQ sequence, as shown in Figure 1 . reading frame under the control of a subgenomic promoter sequence that contains Wuhan sequence 129 S with a diproline (pp) mutation and a QQAQ furin cleavage site sequence.

The RNA is generated by T7 promoter-mediated in vitro transcription using a linearized DNA 132 template. In vitro transcription is performed using an in house-optimized protocol 12,30,31 using T7 133 polymerase, RNase inhibitor, and pyrophosphatase enzymes. DNA plasmid is digested with 134

DNase I and the RNA is capped by vaccinia capping enzyme, guanosine triphosphate, and S-135 adenosyl-methionine. RNA is then purified from the transcription and capping reaction 136 components by chromatography using a CaptoCore 700 resin (GE Healthcare) followed by 137 diafiltration and concentration using tangential flow filtration into 10 mM Tris buffer. The RNA 138 material is terminally filtered with a 0.22 µm polyethersulfone filter and stored at -80 o C until use. 139

The RNA-stabilizing nanostructured lipid carrier (NLC) is comprised of particles with a 140 hybrid liquid and solid oil core, which provides colloidal stability 32 Non-ionic hydrophobic and 141 hydrophilic surfactants help maintain a stable nanoparticle droplet, while a cationic lipid provides 142 the positive charge for electrostatic binding of RNA. That binding on the surface of the 143 nanoparticles protects RNA from degradation by RNases and allowing delivery to cells that will 144 express the S antigen. 145 NLC is manufactured by mixing the lipids in an oil phase, dissolving the Tween 80 in citrate 146 buffer aqueous phase, and homogenizing the two phases by micro-fluidization. The resulting 147 emulsion is sterile-filtered and vialed, and reconstituted in an appropriate buffer before use. 148

The design of vaccination study performed using CD-1 mice is shown in Figure 2 . On the final day of each study, blood was collected via the submandibular vein from 169 isoflurane-anesthetized mice for isolation of sera using a microtainer tube and then mice were 170 euthanized for collection of spleens. Spleens were removed from each mouse and placed in 5 mL 171 of sterile media (RPMI/HEPES/Pen/Strep/10% FBS). Splenocytes were isolated 33 within 2 hours 172 of collection and used fresh or frozen for later analysis. 173

ICS assays were performed using 10 6 live splenocytes per well in 96-well U-bottom plates. 175

Splenocytes in RPMI media supplemented with 10% FBS were stimulated by the addition of pools 176 of overlapping peptides spanning the SARS-CoV-2 S protein (both wild type, wt, or Delta 177 sequence) or N antigens at 2 µg/mL/peptide for 6 h at 37°C in 5% CO2, with protein transport 178 inhibitor, GolgiStop (BD) added two hours after initiation of incubation. The S peptide pool (wild 179 type, JPT Cat #PM-WCPV-S-1; Delta, JPT cat# PM-SARS2-SMUT06-1) is a total of 315 spike 180 peptides split into two pools, S1 and S2, comprised of 158 and 157 peptides each. The N peptide 181 pool (JPT; Cat # PM-WCPV-NCAP-1) was also used to stimulate cells. A SIV-Nef peptide pool 182 (BEI Resources) was used as an off-target negative control. Stimulated splenocytes were then 183 stained with a fixable cell viability stain (eBioscience™ Fixable Viability Dye eFluor™ 506 Cat# 184 65-0866-14) followed by the lymphocyte surface markers CD8b and CD4, fixed with CytoFix 185 (BD), permeabilized, and stained for intracellular accumulation of IFN-γ, TNF-α and IL-2. 186

Fluorescent-conjugated anti-mouse antibodies used for labeling included CD8b antibody (clone 187 H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), TNF-α (clone 188 MP6-XT22, BD) and IL-2 (clone JES6-5H4; BD), and staining was performed in the presence of 189 unlabeled anti-CD16/CD32 antibody (clone 2.4G2; BD). Flow cytometry was performed using a 190

Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software. 191

ELISpot assays were used to detect cytokines secreted by splenocytes from inoculated mice. 193

Fresh splenocytes were used on the same day as harvest, and cryopreserved splenocytes containing 194 lymphocytes were used the day of thawing. The cells (2-4 x 10 5 cells per well of a 96-well plate) 195

were added to the ELISpot plate containing an immobilized primary antibody to either IFN-g or 196 IL-4 (BD Cat# 551881 and BD Cat# 551878, respectively), and were exposed to various stimuli 197 (e.g. control peptides SIV and ConA, S-WT and N peptides pools -see catalog numbers above) at 198 a concentration of 1-2 µg/mL peptide pools for 36-40 hours. After aspiration and washing to 199 remove cells and media, extracellular cytokine was detected by a biotin-conjugated secondary 200 antibody to cytokine conjugated to biotin (BD), followed by a streptavidin/horseradish peroxidase 201 conjugate was used detect the biotin-conjugated secondary antibody. The number of spots per well, 202 or per 2-4 x 10 5 cells, was counted using an ELISpot plate reader. Quantification of Th1/Th2 bias 203 was calculated by dividing the IFN-g spot forming cells (SFC) per million splenocytes with the 204 IL-4 SFC per million splenocytes for each animal. 205

For IgG antibody detection in inoculated mouse sera and lung homogenates, ELISAs for 207 spike-binding (including S1 Delta) and nucleocapsid-binding antibodies and IgG subclasses 208 (IgG1, IgG2a, IgG2b, and IgG3) were used. A microtiter plate was coated overnight with 100 ng 209 of either purified recombinant SARS-CoV-2 S-FTD (FL S with fibritin trimerization domain, 210 constructed and purified in-house by ImmunityBio), purified recombinant Spike S1 domain 211 (S1(wt)) (Sino; Cat # 40591-V08B1), purified recombinant Delta variant Spike S1 domain 212 (S1(Delta)) (Sino; Cat # 40591-V08H23), or purified recombinant SARS-CoV-2 nucleocapsid (N) 213 protein (Sino; Cat # 40588-V08B) in 100 µL of coating buffer (0.05 M Carbonate Buffer, pH 9.6). 214

The wells were washed three times with 250 µL PBS containing 1% Tween 20 (PBST) to remove 215 unbound protein and the plate was blocked for 60 minutes at room temperature with 250 µL PBST. For mice that lack anti-S and/or anti-N specific IgG responses, Th1/Th2 ratio was not calculated. 239

Conversely, some responses, particularly for anti-N responses in IgG2a and IgG2b (both Th1 240 biased subclasses), were above the limit of quantification with OD values higher than those 241 observed in the standard curve. These data points were reduced to values within the standard curve, 242 and thus will reflect a lower Th1/Th2 bias than would otherwise be reported. 243

Serial dilutions were prepared from each serum sample, with dilution factors ranging from 245 400 to 6,553,600 in 4-fold steps. These dilution series were characterized by whole IgG ELISA 246 assays against both recombinant S1(wt) and recombinant S1(Delta), as described above. Half 247 maximal response values (Ab50) were calculated by non-linear least squares fit analysis on the 248 values for each dilution series against each recombinant S1 in GraphPad Prism. Serum samples 249 from mice without anti-S responses were removed Ab50, µg IgG/mL sera, and endpoint titer 250 analyses and reported as N/D on the graphs. Endpoint titers were defined as the last dilution with 251 an absorbance value at least 3 standard deviations higher than the standard deviation of all readings 252 from serum of untreated animals (n = 32 total negative samples). Quantitative titration values (µg 253 IgG/mL sera) were calculated against a standard curve as described above. 254

SARS-CoV-2 pseudovirus neutralization assays were conducted on immunized mouse serum 256 samples using procedures adapted from Crawford et al., 2020. 34 In brief, lentiviral pseudoviruses 257 expressing SARS-CoV-2 spike protein variants were prepared by co-transfecting HEK293 cells 258 Neutralization curves were fit with a four-parameter sigmoidal curve which was used to calculate 274

All statistical analyses were performed and graphs generated used in figures were generated 277 using GraphPad Prism software. Statistical tests for each graph are described in the figure legends. 278

Statistical analyses of Endpoint Titer for anti-S1 IgG (Figure 4) 

Mice receiving the SASA S vaccine in any homologous or heterologous vaccination regimen 284 had the highest levels of anti-full length S(wt) (FL S) IgG2a and 2b as determined by OD at 490 285 nm in ELISA (Fig. 3A) . As expected, only mice receiving the N antigen generated anti-N IgG 286 (also determined by OD at 490 nm in ELISA), which was similar for all groups receiving an N-287 containing antigen (Fig. 3B) by AdS+ N homologous, prime, or boost vaccination. Determination 288 of the IgG1/IgG2a + IgG2b + IgG3 ratio using ng amounts calculated from the OD reading (see 289

Methods) revealed responses were highly T helper cell 1 (Th1)-biased, with all calculated values 290 being greater than one (Fig. 3C) . 291 292

show

IgG3 subtypes represented by OD at 490 nm from ELISA of sera are shown. Statistical analyses 295 performed using one-way ANOVA and Tukey's post-hoc comparison of all groups to all other 296 groups with the exception of comparison to the SASA S > group t SASA S hat did not receive an 297 N antigen for anti-N IgG; where *p ≤ 0.05, **p < 0.01, ***p < 0.001, and ****p<0.0001. (C) The 298

IgG1/IgG2a+IgG2b+IgG3 ratio calculated using the ng equivalents for each is shown with a 299 dashed line at 1. Values > 1 reflect Th1 bias. The number (n) of animals in which the ratio was not 300 determined due to very low antibody levels is shown below the x-axis for each group. The 301

homologous SASA S group did not receive an N antigen. Data graphed as the mean and SEM. 302

The legend in C applies to all figure panels.

Humoral responses against wildtype and Delta S1 were similar in all SASA S groups 305

To assess serum antibody production specific for delta B.1.617.2 variant as compared to 306 wild type S, an ELISAs were performed using either the wt or B.1.617.2 sequence S1 domain of 307 S, which contains the RBD. 308

Vaccine regimens including the SASA S vaccine elicited the highest anti-S1(wt) and 309 S1(Delta) responses as represented by the Ab50, µg IgG/mL, and endpoint titers (Fig. 4A, B , and 310 C, respectively). Four of seven AdS+N homologous vaccinated mice had serum IgG levels against 311 these antigens that were below the level of detection. Overall, the mean antibody titers for SASA 312 S homologous and SASA S > AdS+N groups were highest. For Ab50 and µg IgG/mL (Fig. A and  313 B) statistical comparison of the AdS+N group to other groups was not performed because of the 314 presence of values below the LOD in the AdS+N group. For endpoint titer (Fig. 4C) , the only 315 significant difference was observed between AdS+N homologous versus SASA S homologous 316 vaccination for anti-S1(delta) IgG, with the caveat that for this statistical analysis, serum values of 317 200 for those animals with IgG below the LOD of 400 were used. 318 319 Fig. 4 Wildtype and B.1.617.2 'Delta' S1-specific IgG endpoint titers. Levels of anti-S1(wt) and -320

Delta S1 IgG are shown by (A) Ab50 reciprocal dilution, (B) µg/mL sera, and (C) endpoint titer 321 reciprocal dilution. Values were below the level of detection in 4 of 7 AdS+N homologous group 322 mice. Statistical analyses performed using one-way ANOVA and Tukey's post-hoc comparison of 323 all groups for anti-S1 (WT) or -S1 (Delta) for (C) only where * p = 0.0333; sera without detectable 324 levels of anti-S1 IgG were assigned a value of 200, one-half the Limit of Detection (LOD) of 400. 325

Data graphed as the mean and SEM. The legend in C applies to all figure panels. 326 327 328

Significantly higher percentages of CD4+ T-cells from SASA S > AdS+N group mice secreted 330 IFN-g alone, IFN-g and tumor necrosis factor-a (TNF-a), or IFN-g, TNF-a, and interleukin-2 (IL-331 2) as detected by intracellular cytokine staining (ICS) in response to S(wt) peptides as compared 332 to both the AdS+N or SASA S homologous groups (Fig. 5A, C, and D) . Additionally, the mean 333 percentages were signficantly higher than that of the AdS+ N > SASA S group. 334

The enhancement of cytokine production by AdS+N boost of an SASA S prime was even 335 more pronounced for CD8+ T cells (Fig. 5B , D, and F). Cytokine production was significantly 336 higher in the SASA S > AdS+N group compared to both the homologous vaccination groups as 337 well as the AdS+N > SASA S group. 338

As expected, only T cells from mice receiving vaccination regimens that included delivery of 339 the N antigen by the AdS+N vaccine produced cytokines in response to N peptide stimulation. CD4+ and CD8+ T cells show similar levels of IFN-g production in ICS in response to either 355 S(wt) or S(Delta) sequence peptides ( Fig. 6A and B, respectively) . Patterns of CD4+ and CD8+ 356 T-cell stimulation by S protein peptides between the vaccination regimens were also similar 357 between the S(wt) and S(Delta) peptides. Compared to the untreated control, the significance of 358 the increase in IFN-g production was again the highest for the SASA S > AdS+N group for both 359 CD4+ and CD8+ T cells, and in response to either S(wt) or S(Delta) peptides. Numbers of IFN-g-secreting splenocytes were the highest from mice receiving SASA S > 369

As shown in Figure 7A , ELISpot detection of cytokine secretion in response to the S peptide 371 pool revealed that animals receiving heterologous SASA S > AdS+ N vaccination developed 372 significantly higher levels of S peptide-reactive IFN-g-secreting T cells than all other groups 373 except the SASA S homologous group (which had a lower mean). Numbers of IFN-g-secreting T 374 cells in response to the N peptide pool were similar for AdS+N homologous and SASA S > AdS+N 375 groups. T cells from SASA S > SASA S group animals did not secrete IFN-g in response to the N 376 peptide pool, as expected, because the SASA S vaccine does not deliver the N antigen. While the 377 difference was not significant due to individual variation, the mean number of N-reactive 378 stimulated cells secreting IFN-g due to AdS+N > SASA S vaccination was lower than the other 379 groups receiving a vaccine with N. 380

Induction of interleukin-4 (IL-4) secreting T cells was low for all animals in all groups (Fig.  381 7B), therefore the IFN-g/IL-4 ratio was above 1 for all animals for which the ratio could be 382 calculated (Fig. 7C) , reflecting the Th1-bias of all T-cell responses. The immune responses observed in the present study support our hypothesis that heterologous 

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thus the potential to enhance protection against future SARS-CoV-2 variants of concern that could 421 emerge. We note that mean anti-N IgG responses, while not statistically different among groups 422 that received the N antigen (not SASA S homologous) were, as predicted, highest with 423 homologous AdS+N vaccination.