key: cord-0271411-pyts2nsp authors: Jongeneelen, Mandy; Kaszas, Krisztian; Veldman, Daniel; Huizingh, Jeroen; van der Vlugt, Remko; Schouten, Theo; Zuijdgeest, David; Uil, Taco; van Roey, Griet; Guimera, Nuria; Navis, Marjon; Bos, Rinke; le Gars, Mathieu; Sadoff, Jerald; Muchene, Leacky; Juraszek, Jarek; Langedijk, Johannes PM; Vogels, Ronald; Custers, Jerome; Schuitemaker, Hanneke; Brandenburg, Boerries title: Ad26.COV2.S elicited neutralizing activity against Delta and other SARS-CoV-2 variants of concern date: 2021-07-01 journal: bioRxiv DOI: 10.1101/2021.07.01.450707 sha: 1d64aa4a814faa180918d5df079625f45c4a7325 doc_id: 271411 cord_uid: pyts2nsp Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and recently emerging variants with substitutions in the Spike protein have led to growing concerns over increased transmissibility and decreased vaccine coverage due to immune evasion. Here, sera from recipients of a single dose of our Ad26.COV2.S COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants of concern. All tested variants demonstrated susceptibility to Ad26.COV2.S-induced serum neutralization albeit mainly reduced as compared to the B.1 strain. Most pronounced reduction was observed for the B.1.351 (Beta; 3.6-fold) and P.1 (Gamma; 3.4-fold) variants that contain similar mutations in the receptor-binding domain (RBD) while only a 1.6-fold reduction was observed for the widely spreading B.1.617.2 (Delta) variant. In response to the emergence of SARS-CoV-2 in 2019, Janssen Vaccines developed the Ad26.COV2.S COVID-19 vaccine. The vaccine is currently being tested in multiple clinical trials and interim immunogenicity and efficacy data have been reported [1] [2] [3] . These showed high responder rates for both humoral and cellular immunity against the original SARS-CoV-2 variants (WA1/2020, Wuhan-Hu-1), and high vaccine efficacy (>80%) against severe COVID-19, with complete protection against COVID-19 related hospitalization and death across geographies, including South Africa, where >95% of cases from whom sequence information was available, were infected with the Beta variant of concern (VOC) of SARS-CoV-2 (B.1.351 lineage) 1 . The single dose Ad26.COV2.S COVID-19 vaccine was granted emergency use or (conditional) marketing authorization in more than 50 countries and as of July 2021, more than 19 million people have been vaccinated worldwide. We recently demonstrated that the neutralizing activity in serum against the Beta (B. Participants of phase 3 ENSEMBLE trial (VAC31518COV3001, ClinicalTrials.gov number, NCT04505722) were immunized with one dose of 5x10 10 viral particles of replication-incompetent Ad26 vector encoding for the prefusion-stabilized SARS-COV-2 spike protein sequence (Wuhan-Hu-1; GenBank accession no. MN908947). Sera were collected 71 days later (day 71). Study protocols and results have been reported previously 1 . IgG binding to SARS-CoV-2 spike protein was measured by ELISA using a recombinant and stabilized trimeric spike protein antigen based on the Wuhan-Hu-1 SARS-CoV-2 strain (GenBank accession no. MN908947, D614) 5 . The SARS-CoV-2 spike protein antigen (2.0µg/mL) was directly adsorbed on halfarea 96-well OptiPlate (96W HB, Perkin Elmer) microplates for 2h at 37°C in a humidified incubator. Following incubation, plates were washed three times in PBS/0.05% Tween-20 (PBS-T) and blocked with 1% Casein in PBS for 1h at room temperature. Plates were washed with PBS-T after the blocking step, then serum standards (high titer human convalescent and naïve reference sera), control antibodies, and serum samples were serial diluted (2.5-fold) before incubated on the plates for 1h at room temperature. Next, the plates were washed three times with PBS-T then incubated with peroxidase-conjugated Goat anti-Human IgG (Jackson ImmunoResearch) diluted in blocking buffer for 1h at room temperature, washed three times in PBS-T, and developed with detection substrate (Clarity Western ECL peroxide reagent and luminol enhancer, Bio-Rad) for 10min at room temperature and protected from light. Signal was read out on an Envision plate reader (Perkin Elmer) as relative luminescence units (RLUs). Titers are reported as log10 relative potency (log10 RP) compared with a high titer serum sample used as a reference standard, with a lower limit of quantification at 1.218. For measuring the breadth of neutralization against tested SARS-CoV-2 spike variants, SARS-CoV-2 spike-neutralizing antibody titers were measured in psVNA against several SARS-CoV-2 spike variants as described previously 6 Figure 1 ) were introduced using standard molecular biology techniques and confirmed by sequencing. HIV-based lentiviral pseudotyped particles harboring the SARS-CoV-2 Spike protein variants were produced using the ViraPower Lentiviral Expression system (Thermo Fisher Scientific) according to manufacturer's protocol with minor changes. Neutralization assays for the different variants were performed in parallel on Hek293T target cells stably expressing the human ACE2 and human TMPRSS2 genes (VectorBuilder; Cat. CL0015). Cells were pre-seeded in white half-area 96-well tissue culture plates (Perkin Elmer) at a density of 1.5 × 10 4 cells/well. Heat-inactivated serum samples were two-fold serial diluted in duplicates over 10 columns in phenol red free DMEM supplemented with 1% FBS and 1% PenStrep. Serum standards (high titer human convalescent and naïve reference sera), controls (including antibodies), and serial diluted serum samples were incubated at room temperature with an equal volume of pseudovirus particles with titers of ∼1 × 10 5 RLUs luciferase activity. After 1h incubation, the serum-particle mixture was inoculated onto Hek293T.ACE2.TMPRSS2 cells. Luciferase activity was measured 40h after transduction by adding an equal volume of NeoLite substrate (Perkin Elmer) to the wells according to the manufacturer's protocol, followed by readout of RLUs on the EnSight Multimode Plate Reader (Perkin Elmer). SARS-CoV-2 neutralizing titers were calculated in R using a four-parameter curve fit as the sample dilution at which a 50% reduction (IC50) of luciferase readout was observed compared with luciferase readout in the absence of serum ("High Control"). The starting serum sample dilution of 20 was fixed as the limit of detection (LOD). Geometric mean titers, limit of detection, and fold change to B.1 were reported. Statistical comparison of the fold differences relative to B.1 were performed in SAS 9.4 using a paired-sample T-test at a 5% significance level. No adjustment for multiple comparisons were done. The corresponding 95% confidence intervals were also reported. Sera from phase 3 ENSEMBLE trial participants (n=8; from Brazil, RSA, and USA, 71 days post single dose vaccination, age ranging from 47 to 91 years) were evaluated for their ability to bind to B.1 Spike protein in ELISA and for their neutralizing activity against B.1 and variants of concern (listed in Figure 1 (Figure 3) . We recently reported that cellular immunity is conserved against SARS-CoV-2 variants of concern 4 . While we have not yet tested this for the Delta VOC, in silico analysis has demonstrated the preservation of dominant T cell epitopes in this variant (data not shown). Safety and efficacy of single-dose Ad26.COV2.S vaccine against Covid-19 Immunogenicity of the Ad26.COV2.S Vaccine for COVID-19 Interim results of a phase 1-2a trial of Ad26.COV2.S Covid-19 vaccine Immunogenicity of Ad26.COV2.S vaccine against SARS-CoV-2 variants in humans Stabilizing the closed SARS-CoV-2 spike trimer Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Reduced neutralization of SARS-CoV-2 B. 1.617 by vaccine and convalescent serum Effectiveness of COVID-19 vaccines against hospital admission with the Delta (B.1.617.2) variant. Public Health England U.; formal & statistical analysis All authors have read and agreed to the published version of the manuscript We thank all participants of our clinical trials. We thank Lucy Rutten for providing the S-ELISA antigen.