A COVID-19 DNA Vaccine Candidate Elicits Broadly Neutralizing Antibodies against Multiple SARS-CoV-2 Variants including the Currently Circulating Omicron BA.5, BF.7, BQ.1 and XBB

Abstract

Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants currently pose a global challenge to the control of the COVID-19 pandemic. We previously reported a pVAX1-based DNA vaccine candidate, pAD1002, that encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 wildtype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1 RBD-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-γ⁺ cellular responses in BALB/c and C57BL/6 mice. More importantly, pAD1016 vaccination in mice, rabbits and pigs generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These preliminary data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad-spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy of further translational study as a COVID-19 vaccine candidate.

Keywords: COVID-19; DNA vaccine; Omicron; RBD chimera; SARS-CoV-2.

Figure 1

Construction of pAD1016. (A) Schematic diagram comparing the primary structure of secreted polypeptides encoded by pVAX1-based vaccine candidates pAD1002 and pAD1016. (B) qPCR detection of RBD mRNA transcripts in HEK293T cells transiently transfected with pVAX1 or pAD1016. (C) Samples of HEK293T cells transiently transfected with, or without (NC), pVAX-1, pAD1002 or pAD1016 were run SDS-PAGE gel followed by Western blotting using anti-RBD^SARS Abs for detection. Recombinant RBD^SARS was included as control. (D) Secreted recombinant heterodimeric RBD polypeptide in culture supernatant of the transfectant HEK293T cells was quantitated using anti-RBD^SARS Ab-based sandwich ELISA. HRP-labeled anti-RBDWT Ab was employed as detection Ab. Serum samples, collected from BALB/c (E) and C57BL/6 (F) mice after secondary immunization with 20 μg pAD1016 DNA via MAP-1016 administration (MAP), intradermal injection (ID) or intramuscular inoculation followed by EP (IM+EP), were titrated against recombinant RBD^WT in ELISAs. Normal mouse serum (NMS) was included as negative control. Data represent mean ± SD (n = 5 biologically independent samples). The original whole blot and the densitometry readings of the bands in WB shown in Supplementary Figure S3.

Figure 2

SARS-CoV-2-specific T cell responses in pAD1016-vaccinated mice. C57BL/6 mice were IM+EP immunized twice with pVAX1, pAD1002 or pAD1016 (n = 5, 20 μg/dose, fortnight interval) and then sacrificed for spleens 14 days after boost. ELISpot analyses of IFN-γ spot-forming cells (SFC) amongst splenocytes after re-stimulation with, or without (NC), pooled 14-mer overlapping peptides covering the sequences of RBD^SARS (A) or RBD^BA4/5 (B) were performed.

Figure 3

Neutralization antibodies induced by pAD1016 in mice and rabbits. (A) C57BL/6 mice (n = 5) were administered with 2 doses (20 μg/dose, fortnight interval) of pAD1016/IM+EP followed by serum sample collection 14 days after boost. An equal proportion mixture of the serum samples was tested for ability to block mimic infection of ACE2-expressing HEK293T cells by pseudoviruses displaying S protein of SARS-CoV-2 WT, Delta, Omicron BA.2 or BA.4/5 variants. (B) Rabbits (n = 3) administered with 2 doses (500 μg/dose, fortnight interval) of pAD1016/IM+EP followed by serum sample collection 14 days after boost immunization. An equal proportion mixture of the serum samples was tested for ability to block mimic infection of ACE2-expressing HEK293T cells by pseudoviruses displaying S protein of SARS-CoV-2 WT, Delta, Omicron BA.2, BA.4/5 or BF.7. (C) A serum sample from rabbits (equal proportion mixture of 3 rabbits) 14 days after 2 doses of MAP-1016 immunization (500 μg/MAP/dose, fortnight interval) was assayed for ability to block mimic infection of ACE2-expressing HEK293T cells by pseudoviruses displaying S protein of SARS-CoV-2 Omicron BA.7 or XBB. The results are expressed as percent inhibition of infection (left panels) and NT50 titers (right panels). Data are means ± SEM.

Figure 4

Effect of pAD1016 boost to inactivated virus pre-vaccination in mice. C57BL/6 mice (n = 5) were i.m. administered with two doses of inactivated SARS-CoV-2 virus vaccine (60 units/dose), and then boosted with, or without (Inact/Inact), either inactivated SARS-CoV-2 virus vaccine (Inact/Inact/Inact) or 20 μg pAD1016/IM+EP (Inact/Inact/pAD1016). Serum samples, collected 14 days after the secondary, or third, immunization were assayed for ability to block mimic infection of ACE2-expressing HEK293T cells by pseudoviruses displaying S protein of SARS-CoV-2 variants Omicron BA.4/5 (A), BF.7 (B), BQ.1 (C) or Delta (D). Normal mouse serum was included as negative control (NC). The results are expressed as percent inhibition of infection. Data are means ± SEM.

Figure 5

Comparison of AI-modeled molecular structure of the polypeptides encoded by pAD1002, pAD1003, pAD131 and pAD1016. Of the 4 fusion RBD heterodimers, RBD^Beta/BA1 forms “bilateral lung” structure, indicating stable complexation between RBD^BA1 and RBD^Beta, while RBD^SARS/Beta, RBD^SARS/BA1 and RBD^SARS/BA5 are all in free-moving conformation, suggesting a possibility that RBD^SARS can barely complex with RBDs of SARS-CoV-2 variants under physiological conditions.