A Single Vaccine Protects against SARS-CoV-2 and Influenza Virus in Mice

Abstract

The ongoing SARS-CoV-2 pandemic poses a severe global threat to public health, as do influenza viruses and other coronaviruses. Here, we present chimpanzee adenovirus 68 (AdC68)-based vaccines designed to universally target coronaviruses and influenza. Our design is centered on an immunogen generated by fusing the SARS-CoV-2 receptor-binding domain (RBD) to the conserved stalk of H7N9 hemagglutinin (HA). Remarkably, the constructed vaccine effectively induced both SARS-CoV-2-targeting antibodies and anti-influenza antibodies in mice, consequently affording protection from lethal SARS-CoV-2 and H7N9 challenges as well as effective H3N2 control. We propose our AdC68-vectored coronavirus-influenza vaccine as a universal approach toward curbing respiratory virus-causing pandemics. IMPORTANCE The COVID-19 pandemic exemplifies the severe public health threats of respiratory virus infection and influenza A viruses. The currently envisioned strategy for the prevention of respiratory virus-causing diseases requires the comprehensive administration of vaccines tailored for individual viruses. Here, we present an alternative strategy by designing chimpanzee adenovirus 68-based vaccines which target both the SARS-CoV-2 receptor-binding-domain and the conserved stalk of influenza hemagglutinin. When tested in mice, this strategy attained potent neutralizing antibodies against wild-type SARS-CoV-2 and its emerging variants, enabling an effective protection against lethal SARS-CoV-2 challenge. Notably, it also provided complete protection from lethal H7N9 challenge and efficient control of H3N2-induced morbidity. Our study opens a new avenue to universally curb respiratory virus infection by vaccination.

Keywords: COVID-19; SARS-CoV-2 variants; adenoviral vector; influenza; vaccine.

FIG 1

Construction of AdC68-CoV/Flu and verification of immunogen expression. (A) Schematic representation of design of AdC68-CoV/Flu, a vaccine intended to doubly target SARS-CoV-2 and influenza A virus. The fusion immunogen is comprised of three parts: RBD from SARS-CoV-2, HA stalk (HA2) from influenza H7N9, and human ferritin. (B) Verification of immunogen expression. HEK 293T cells were infected with the indicated amount (vps) of AdC68-CoV/Flu or AdC68 viruses in a 6-well plate and harvested 24 h postinfection. Resultant lysates were analyzed by Western blotting using a rabbit antibody specific for S protein of SARS-CoV-2. (C) Assessment of N-glycosylation of RBD. Purified BSA and RBD-ferritin proteins were treated or untreated with a deglycosylation mix under native conditions (+) versus more effectively denaturing conditions (++) and were then subjected to SDS gel electrophoresis. The separated proteins were visualized by Coomassie blue staining. (D) Verification of the structure of immunogen. HEK 293T cells were infected with 10¹⁰ vp of AdC68-CoV/Flu virus in a 6-well plate and harvested 24 h postinfection. Cell lysates were separated on SDS gel under reduced (+DTT) and nonreduced (-DTT) conditions and analyzed by immunoblotting using a rabbit antibody specific for S protein of SARS-CoV-2. The protein band with expected size of trimeric immunogen was marked by dashed rectangle.

FIG 2

Immunogenicity of AdC68-CoV/Flu in mice. (A) Scheme of vaccination and sampling schedule. The mice were intramuscularly administered with AdC68-CoV/Flu or AdC68 in 5 × 10¹⁰ viral particles (vp) per dose following a homologous prime-boost regimen with a 4-week interval. (B) Serum were assessed by ELISA at week 6 post-prime for RBD-specific binding antibodies. (C) Assessment of Th1 or Th2 bias in the immune response. RBD-specific serum antibodies of IgG1 and IgG2a isotypes were determined by ELISA at week 6 post-prime, as their ratio serves as a surrogate measure of Th1/Th2 immune balance. (D) At week 6 post-prime, serum-neutralizing antibody titers against wild-type SARS-CoV-2 were measured by pseudovirus neutralization assays. (E) At week 10 post-prime, serum-neutralizing antibody titers against either wild-type SARS-CoV-2 or its B.1.1.7 and B.1.351 variants were assessed by pseudovirus neutralization assay. (F and G) Assessments of RBD-specific T cell responses. Splenocytes were isolated at week 5 post-prime and in vitro stimulated with 13 peptide pools (15-mer with 11 overlapped amino acids) covering the entire RBD sequence. The resulting IFN-γ secreting cells were quantified by ELISpot, recorded as a total response to the whole peptide pools (panel F) and individual peptide pools (panel G). Empty RMPI1640 complete medium, denoted as R10, was used as a negative control. Titer data are presented as geometric mean titers (GMT) ± geometric standard deviation (GSD); ELISpot counts were expressed as mean ± standard error of the mean. A Mann-Whitney test was performed to analyze differences between experimental groups.

FIG 3

Protective efficacy of AdC68-CoV/Flu against SARS-CoV-2 challenge in hACE2 mice. (A) Experimental schedule. Transgenic hACE2-C57BL/6 mice (n = 8/group) were primed with intramuscular injections of 5 × 10¹⁰ vp of either AdC68 (sham) or AdC68-CoV/Flu on week 0, and 4 weeks later were boosted with 1 × 10¹¹ vp of the same vaccine via both intramuscular and intranasal routes. This was followed by an intranasal challenge of 1 × 10⁴ PFU of SARS-CoV-2 (CHN/Shanghai_CH-02/2020) on week 7. (B) Lung viral loads on day 3 postinfection (dpi) were determined by quantitative RT-PCR of viral N transcripts in lung homogenates, which were plotted as log₁₀ copies per mL. (C) Representative images of H&E-stained lung sections from AdC68- and AdC68-CoV/Flu-treated mice on 3 dpi. Scale bar, 200 μm. (D and E) Protection evaluated by % body weight change (panel D) and % survival (panel E). Between-group comparisons were conducted by Mann-Whitney tests.

FIG 4

Influenza-targeting immunity raised by AdC68-CoV/Flu and the protection afforded against H7N9 and heterologous H3N2 challenges in mice. (A) Experimental schedule used for evaluating AdC68-CoV/Flu-mediated H7N9 protection. ICR mice (n = 5/group) were intramuscularly vaccinated with 5 × 10¹⁰ vp of either AdC68 (sham) or AdC68-CoV/Flu at weeks 0 (prime) and 4 (boost), followed by intranasal challenge with 1 × 10⁵ TCID₅₀ of A/Shanghai/4664T/2013 (H7N9) influenza virus at week 7. (B) Serum titers of H7 HA-specific binding antibodies at week 6 post-prime, determined by ELISA. (C and D) Protection evaluated by % body weight change (panel C) and % survival (panel D). (E) Experimental schedule used for evaluating protective efficacy of AdC68-CoV/Flu against H3N2 challenge. BALB/c mice (n = 5/group) were subjected to intramuscular vaccination with 5 × 10¹⁰ vp of either AdC68 or AdC68-CoV/Flu at weeks 0 (prime) and 4 (boost), followed by intranasal challenge with 5 × 10⁵ TCID₅₀ of A/Hong Kong/68 (H3N2) influenza virus H3N2 at week 8. (F) Serum titers of H3 HA-specific binding antibodies at week 6 post-prime, determined by ELISA. (G) Body weight change over time. Titer data are presented as geometric mean titers (GMT) ± geometric standard deviation (GSD). Between-group comparisons were conducted by Mann-Whitney tests.