key: cord-1009935-4edmfuuh
authors: Swart, Maarten; de Wilde, Adriaan; Schmit-Tillemans, Sonja; Verspuij, Johan; Daal, Chenandly; Choi, Ying; Perkasa, Aditya; Kourkouta, Eleni; Tahiri, Issam; Mulders, Michel; Gil, Ana Izquierdo; Muchene, Leacky; Juraszek, Jarek; Vellinga, Jort; Custers, Jerome; Bos, Rinke; Schuitemaker, Hanneke; Wegmann, Frank; Roozendaal, Ramon; Kuipers, Harmjan; Zahn, Roland
title: Immunogenicity of an Ad26-based SARS-CoV-2 Omicron Vaccine in Naïve Mice and SARS-CoV-2 Spike Pre-immune Hamsters
date: 2022-03-04
journal: bioRxiv
DOI: 10.1101/2022.03.04.482636
sha: 026d9ef986095fa552d14babd0221ca590b8c6b9
doc_id: 1009935
cord_uid: 4edmfuuh

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant sparked concern due to its fast spread and the unprecedented number of mutations in the spike protein that enables it to partially evade spike-based COVID-19 vaccine-induced humoral immunity. In anticipation of a potential need for an Omicron spike-based vaccine, we generated an Ad26 vector encoding an Omicron (BA.1) spike protein (Ad26.COV2.S.529). Ad26.COV2.S.529 encodes for a prefusion stabilized spike protein, similar to the current COVID-19 vaccine Ad26.COV2.S encoding the Wuhan-Hu-1 spike protein. We verified that spike expression by Ad26.COV2.S.529 was comparable to Ad26.COV2.S. Immunogenicity of Ad26.COV2.S.529 was then evaluated in naïve mice and SARS-CoV-2 Wuhan-Hu-1 spike pre-immunized hamsters. In naïve mice, Ad26.COV2.S.529 elicited robust neutralizing antibodies against SARS-CoV-2 Omicron (BA.1) but not to SARS-CoV-2 Delta (B.1.617.2), while the opposite was observed for Ad26.COV2.S. In pre-immune hamsters, Ad26.COV2.S.529 vaccination resulted in robust increases in neutralizing antibody titers against both SARS-CoV-2 Omicron (BA.1) and Delta (B.1.617.2), while Ad26.COV2.S vaccination only increased neutralizing antibody titers against the Delta variant. Our data imply that Ad26.COV2.S.529 can both expand and boost a Wuhan-Hu-1 spike-primed humoral immune response to protect against distant SARS-CoV-2 variants.

In response to the SARS-CoV-2 (COVID-19) pandemic, multiple mostly spike-based vaccines 32 were rapidly and successfully developed. These vaccines showed high efficacy against COVID-33 19 and have been deployed worldwide. Janssen developed the Ad26.COV2.S vaccine, which is 34 a replication-incompetent human adenovirus type 26 (Ad26) vector 1 encoding a stabilized pre-35 fusion SARS-CoV-2 spike protein based on the Wuhan-Hu-1 isolate 2 . A phase 3 trial 36 demonstrated that Ad26.COV2.S was 74.6% efficacious at preventing severe-critical COVID-19 3 . 37

The Ad26.COV2.S COVID-19 vaccine was granted emergency use authorization in the US and 38 (conditional) marketing authorization in the European Union; in more than 50 other countries. 39

Several rapidly spreading SARS-CoV-2 variants have evolved since the initial introduction 40 of the virus into humans. The Beta (B.1.351) and Delta (B.1.617.2) variants of concern (VOC) 41

were initially thought to potentially evade vaccine elicited immunity. However, COVID-19 vaccine 42 efficacy was largely maintained 3-8 . The Delta VOC obtained virtually worldwide dominance until it 43 was replaced by the Omicron variant, BA.1 (initially named B. 1.1.529) . After its first reporting in 44 November 2021, BA.1 was instantly declared a VOC due to its rapid spread and the unparalleled 45 number of mutations in the spike protein. BA.1 carries 15 mutations in its receptor binding domain 46 (RBD), which is an immunodominant target for neutralizing antibodies 9 , raising the possibility of 47 Female BALB/c mice aged 8-10 weeks at the start of study were purchased from Charles 104 River Laboratories (Germany). Mice were immunized intramuscularly with 100 μl (50 μl per 105 hindleg) vaccine under general anesthesia with isoflurane. Blood samples were collected via 106 submandibular bleeding. 107

Female Syrian golden hamsters (Mesocricetus auratus), strain RjHan:aura, aged 9-11 108 weeks at the start of the study, were purchased from Janvier Labs (France). Assays were performed on HEK293T target cells stably expressing the human 122 angiotensin-converting enzyme 2 (ACE2) and human transmembrane serine protease 2 (TMPRSS2) genes (VectorBuilder, Cat. CL0015). The cells were seeded in white half-area 96-124 well tissue culture plates (Perkin Elmer) at a density of 1.5 x 10 4 cells/well. 125 Two-fold serial dilutions were prepared from heat-inactivated serum samples in phenol 126 red free Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 1% FBS and 1% 127

PenStrep. The serially diluted serum samples were incubated at room temperature with an equal 128 volume of pseudovirus particles with titers of approximately 1 x 10 5 Relative Luminescence Units 129 (RLU) luciferase activity. After one hour incubation, the serum-particle mixture was inoculated 130 onto HEK293T.ACE2.TMPRSS2 cells. Luciferase activity was measured 40 h after transduction 131 by adding an equal volume of NeoLite substrate (Perkin Elmer) to the wells according to the 132 manufacturer's protocol, followed by read out of RLU on the EnSight Multimode Plate Reader 133 (Perkin Elmer). SARS-CoV-2 neutralizing titers were calculated using a four-parameter curve fit 134 as the sample dilution at which a 50% reduction (N50) of luciferase readout was observed 135 compared with luciferase readout in the absence of serum (High Control). The starting serum 136 sample dilution of 20 was fixed as the lower limit of detection (LLOD). 137 138

Statistical comparisons were performed in SAS 9.4 using a paired-sample t-test from an ANOVA. 140

If titers were censored at LLOD, then a Tobit z-test from a Tobit ANOVA was used instead. If a 141 vaccine-dose had more than 50% censored measurements, the non-parametric Mann-Whitney 142 U-test was used instead. No adjustments for multiple comparisons were done. 143 144

In-vitro characterization of spike expression by Ad26.COV2.S.529 146

Ad26.COV2.S.529 vector spike expression and antigenicity were characterized in vitro and 147 compared to Ad26.COV2.S. Spike protein expression was evaluated after transduction of A549 148 cells using a quantitative cell-based ELISA with CV3-25 and ACE2-Fc. CV3-25 is an antibody that 149 binds to the stem region of the SARS-CoV-2 spike S2 21 , which is conserved between the Wuhan-150

Hu-1 and BA.1 spike protein and ACE2-binding affinity is reported to be similar between Wuhan-151 Hu-1 Spike and Omicron spike 25, 26 . Here, we show that CV3-25-binding to the spike protein 152 expressed after transduction of A549 cells with Ad26.COV2.S or Ad26.COV2.S.529 was 153 comparable (Figure 1A) . Similarly, ACE2-Fc fusion protein binding to both spike proteins was 154 comparable (Figure 1B) . Naïve mice were immunized with 10 8 , 10 9 , 10 10 viral particles (vp) of Ad26.COV2.S, 165

Ad26.COV2.S.529 or 10 10 vp Ad26.Empty mock control vector. SARS-CoV-2 spike neutralization titers were evaluated using a psVNA assay in sera collected 4 weeks after immunization. We 167 focused our analysis on the two most recent and prevalent VOCs worldwide: Delta (B.1.617.2) 168 and Omicron (BA.1). A single immunization with Ad26.COV2.S.529 induced dose-dependent 169

Omicron spike neutralizing antibodies that were significantly higher than after vaccination with 170

Ad26.COV2.S at all doses tested (Figure 2A) . In contrast, Omicron spike neutralizing titers 4 171 weeks after vaccination with Ad26.COV2.S were comparable to animals vaccinated with 172

Ad26.Empty. While Ad26.COV2.S induced robust dose-dependent Delta spike neutralizing 173 antibody titers, Delta titers in animals vaccinated with 10 8 and 10 9 vp Ad26.COV2.S.529 were in 174 the same range as after vaccination with Ad26.Empty (Figure 2A) . Only the highest dose of 175

Ad26.COV2.S.529 tested (10 10 vp) induced statistically significantly Delta spike neutralization 176 titers compared with Ad26.Empty, albeit at lower levels compared to 10 10 vp Ad26.COV2.S. 177

As an increasing part of the population acquired pre-existing immunity either by infection 178 or vaccination, we also evaluated the immunogenicity of Ad26.COV2.S.529 in hamsters with pre- robust Omicron (BA.1) spike neutralizing titers in pre-immune hamsters, while Ad26.COV2.S did 234 not. The capacity of Ad26.COV2.S.529 to elicit antibodies with the ability to neutralize Omicron in 235 hamsters that were pre-exposed to the spike protein of the SARS-CoV-2 Wuhan-Hu-1 strain 236 indicates either boosting of low level pre-existing common neutralizing epitope specific memory 237 B cells or de-novo induction of B cells that produce antibodies that can neutralize Omicron. 238

Our data seem to contrast data from an Omicron-based mRNA vaccine that provided 239 similar neutralizing antibody levels against Omicron as compared to the original Wuhan-Hu-1-240 based mRNA vaccine in nonhuman primates (NHP) that were previously immunized with 2 doses 241 of the original mRNA vaccine 35 . The Omicron-based mRNA vaccine did however elicit higher 242

Omicron-neutralizing titers in pre-immune mice as compared to the original mRNA vaccine 31 . 243 Nevertheless, we have previously shown in pre-immune NHPs, that a heterologous booster 244 vaccination with an Ad26 vector-based vaccine candidate expressing a stabilized Beta-based 245 spike protein (Ad26.COV2.S.351) >7 months after Ad26.COV2.S vaccination elicited 2.9-fold 246 higher Beta psVNA titers than homologous vaccination with the Ad26.COV2.S 36 . It is currently 247 unclear if these differences are related to the peak magnitude of immune responses elicited by 248 previous immunizations, the number of pre-immunizations with the original vaccine prior to 249 vaccination with the Omicron-based vaccine, the vaccine platform or the experimental animal 250 models used. 251

Ad26.COV2.S.529 vaccination resulted in similar Delta spike neutralization titers as 252

Ad26.COV2.S in pre-immune hamsters, but Delta neutralizing titers were undetectable or very 253 low in Ad26.COV2.S.529 vaccinated naïve mice. The latter is consistent with data from naïve 254 animals vaccinated with Omicron-specific mRNA vaccines 30-33 and also in Omicron-infected 255 hamsters 37 where Delta spike neutralizing antibodies were undetectable. This suggests that an 256 immunization with a heterologous spike protein as encoded by Ad26.COV2.S.529 induces a more 257 broadly neutralizing antibody response in animals with pre-existing immunity to ancestral Wuhan-258

Hu-1 spike than a homologous spike protein immunization. Ongoing studies in non-human 259 primates and potential future studies in humans may confirm these findings. 

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Ad26.COV2.S elicited neutralizing activity against Delta and other 330 SARS-CoV-2 variants of concern

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