An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques

Abstract

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 10¹⁰ viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.

Fig. 1. Construction and characterization of Ad5-S-nb2.

a Schematic diagram of the genome of Ad5-S-nb2 and the coding sequence for SARS-CoV-2 S protein. b Western blot analysis of the expression of S protein in HEK293 cells transfected with plasmids encoding an original S sequence (pGA1-S-nb1, 4 μg per well) or a codon-modified S sequence (pGA1- S-nb2 1#: 4 μg per well; pGA1- S-nb2 2#: 2 μg per well). A pGA1-empty plasmid was used as the negative control. A purified S protein with the transmembrane domain truncated (S_ΔTM) was used as the positive control. c Western blot analysis of the expression of S protein in HEK293 cells infected with Ad5-S-nb2. Ad5-S-nb2 1#, 0.2 TCID50 per cell; Ad5-S-nb2 2#, 0.05 TCID50 per cell. S_ΔTM protein and HEK293 cells infected with Ad5-empty were examined in parallel as the positive and negative controls, respectively. The samples were derived from the same experiment and the blots were processed in parallel. d Immunofluorescence analysis of the expression of S protein in A549 cells mediated by Ad5-S-nb2. A549 cells were infected with Ad5-S-nb2 or Ad5-empty at 0.2 TCID50 per cell. Twenty-four hours later, cells were labeled with a human monoclonal antibody against S protein and then with an Alexa Fluor 488-conjugated mouse anti-human antibody. The cells were observed under a fluorescence microscope. Scale bar = 50 μm. For b and c, two independent experiments were carried out with similar results. For d, one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

Fig. 2. The immunogenicity of Ad5-S-nb2 in mice.

a Schematic diagram of the schedule, dosage, and delivery routes of mouse experiment. Eight-week-old BALB/c mice (n = 5 per group) were vaccinated with Ad5-S-nb2 using the indicated dosage through an IM or IN route. The gray blood drop symbols indicate the time points at which the serum samples were collected. Twenty-eight days after the initial vaccination, the mice were sacrificed. b The IgG antibody response in the sera of mice that received IM vaccination. c The IgA antibody response in the BALFs of mice that received IM vaccination. d The IgG antibody response in the sera of mice that received IN vaccination. e The IgA antibody response in the BALFs of mice that received IN vaccination. The endpoint titers of IgG antibodies (b and d) and IgA antibodies (c and e) against the RBD, S2, and S protein are shown. f The titers of neutralizing antibodies in mice sera. g The titers of neutralizing antibodies in mice BALFs. The sera (f) or BALFs (g) from each group of mice (n = 5) were pooled and assessed by S-pseudotyped reporter lentivirus. h, j IFN-γ-secreting cells in the spleens of mice that received IM (h) or IN vaccination (j). Splenic lymphocytes from each group of mice (n = 5) were pooled and stimulated with two peptide pools corresponding to the S1 and S2 region. Shown are the number of spot-forming cells (SFCs) in one million splenic lymphocytes. For b–e, each circle represents an individual mice, and the error bars indicate the means ± standard derivations (SD). For f–i, each bar reflects the mean value of three technical replicates. Data are one representative result of two independent experiments. Comparisons were performed by Student’s t-test (unpaired, two-tailed). Source data are provided as a Source Data file.

Fig. 3. The immunogenicity of Ad5-S-nb2 in Chinese rhesus macaques.

a Schematic diagram of the vaccination and challenge studies in rhesus macaques. Three groups of macaques (n = 4 per group) were vaccinated via an IM injection of 1 × 10¹⁰ vp or 1 × 10¹¹ vp Ad5-S-nb2 or via an IN and oral inoculation of 5 × 10¹⁰ vp Ad5-S-nb2 each. The control groups include macaques (n = 2) IM injected with 1 × 10¹¹ vp Ad5-empty and non-vaccinated macaques (n = 6). On days 0, 12, 18, and 24 after vaccination, the serum samples and PBMCs were collected. On day 30 (Group 1 and 2) or 56 (Group 3) after vaccination, macaques were challenged. On day 7 or 10 after challenge, macaques were euthanatized. The gray inverted triangles indicate the time points of vaccination or challenge, whereas the gray blood drop symbols indicate the time points at which the serum samples were collected. b The kinetics of IgG response to the S protein in the sera. The endpoint IgG titers are shown. The overlapped data points represent the same values. Comparisons between different time points were performed by Student’s t-test (paired, one-tailed). c The antigen recognition profiles of macaque immune sera. Macaque immune sera collected on day 24 were assessed for the IgG antibodies against the RBD, S2, and S protein by enzyme-linked immunosorbent assay (ELISA). d IFN-γ-secreting cells in the PBMCs of rhesus macaques. PBMCs isolated on day 18 were stimulated with two peptide pools corresponding to S1 and S2, respectively. Shown are the number of SFCs in one million PBMCs. All the data points represent the mean values of two technical replicates. Source data are provided as a Source Data file.

Fig. 4. Ad5-S-nb2 protects against SARS-CoV-2 infection in rhesus macaques.

a The viral loads in the pharyngeal swabs of non-vaccinated macaques. b The viral loads in the pharyngeal swabs in macaques at 4 weeks after IM vaccination with 1 × 10¹¹ vp Ad5-S-nb2. c The viral loads in the pharyngeal swabs in macaques at 4 weeks after IN vaccination with 5 × 10¹⁰ vp Ad5-S-nb2. d The viral loads in the pharyngeal swabs in macaques at 8 weeks after IM vaccination with 1 × 10¹⁰ vp Ad5-S-nb2. The genome copy numbers in the elution of the pharyngeal swabs were determined by quantitative reverse transcription PCR (qRT-PCR). The limit of detection was 200 copies ml⁻¹ and marked by the dot line. The data points were expressed as the mean of two technical replicates. Macaques C3, 116004, 134018, 063585, and 080066 were euthanatized on day 7 after challenge and were marked by hash symbol. Other macaques were euthanatized on day 10 after challenge. Macaques C1, C2, D1 and D2 were subsequently utilized for another study. e The viral loads calculated based on AUC in the pharyngeal swabs in macaques after challenge. Blue circles, macaques that received 1 × 10¹¹ vp Ad5-S-nb2 (IM); Dark cyan circles, macaques that received 1 × 10¹⁰ vp Ad5-S-nb2 (IM); Red circles, macaques that received mucosal vaccination (IN). Black circles, non-vaccinated macaques. Black lines reflect the mean AUC. Comparison between vaccinated (n = 9) and non-vaccinated (n = 5) macaques was conducted using Student’s t-test (unpaired, two-tailed). f–i The neutralizing antibody titers against SARS-CoV-2 before and after challenge in non-vaccinated macaques (f), high-dose IM vaccinated macaques (g), IN vaccinated macaques (h), and low-dose IM vaccinated macaques (i). The sera were examined by PRNT using SARS-CoV-2 (strain 2019-nCoV-WIV04). Source data are provided as a Source Data file.

Fig. 5. Viological and histopathological analysis of macaque airway tissues.

a The viral loads in the nine anatomic locations of macaque airway. The tissues of trachea, left and right bronchus, and the upper, middle, and lower locations of the left and right lung were collected at euthanasia. The viral genomes in the tissue homogenates were assessed by qRT-PCR. All the data points represent the mean values of two technical replicates. The dot line indicates the limit of detection (1000 copies per g tissue). b–f Histopathological changes in the lungs of SARS-CoC-2 challenged non-vaccinated macaques (b) and vaccinated macaques (d–f), and an unchallenged macaque IM vaccinated with 1 × 10¹⁰ vp Ad5-S-nb2 (c). The lung tissue sections were stained with hematoxylin and eosin (H&E). One representative graph from each macaque is shown. Scale bars are 50 μm and indicated in each panel. Source data are provided as a Source Data file.