Construction of ORFV D1701-VrV-based SARS-CoV-2 recombinants expressing spike protein alone or in combination with nucleocapsid protein
We used the D1701-VrV ORFV vector31, 32 to generate two SARS-CoV-2 vaccine candidates as described previously (Fig. 1A).26 The full-length spike protein from of the ancestral SARS-CoV-2 (Wuhan) genomic sequence33 was inserted into the vegf-e locus under the control of the early Pvegf promotor of D1701-VrV ORFV in both recombinants. The spike protein sequence included mutations D614G, mutation K986P and V987P for proline stabilization and a furin cleavage site deletion (aa 682–685 RRAR to GSAS.)34 The nucleocapsid gene was inserted in the D (Del2) locus under control of an artificial early P7 promotor, generating a double transgene-containing recombinant (ORFV-S/N) (Fig. 1A). The early Pvegf and P7 promotors initiated antigen expression encoded in the virus in the cytoplasm of the infected cells. We confirmed the integrity, correct and stable insertion of transgenes by vegf-, Del2- and transgene-specific PCR, and gene sequencing. We used an empty D1701-VrV ORFV vector as a control (ORFV-Mock).
We confirmed spike protein expression from ORFV-S and ORFV-S/N by flow cytometry 20 hours after infecting Vero cells using multiplicity of infection (MOI) of 1 by cell surface staining (Fig. 1B; left panel) and intracellular staining (Fig. 1B; middle panel). The mean fluorescence intensities were comparable for both recombinants. We also verified intracellular nucleocapsid protein production by flow cytometry in Vero cells infected with the ORFV-S/N recombinant (Fig. 1B; right panel) and confirmed the correct transgene expression by western blotting using Vero cells harvested 48 hours after infection (MOI 1) and detected specific bands (180 kDa) in cell lysates using antibodies against S1 and S2 subunits of spike protein (Fig. 1C), as well as against the nucleocapsid protein (46 kDa) (Fig. 1C).
Mono- and multi-antigenic ORFV recombinants induce robust and long-lasting T H 1-biased antibody- and cellular immune responses against inserted transgenes in mice
The immunogenicity of both ORFV-based vaccine candidates was evaluated in CD-1 mice after two intramuscular (i.m.) injections with a 21-days interval using 107 PFU. Mice immunized with ORFV-Mock recombinant were used as controls (Fig. 2A). After a single injection, both ORFV vaccines induced similar levels of RBD-specific binding antibodies with geometric mean endpoint titers (GMT) of 3.9×105 and 2.3×105, respectively, 3-weeks after vaccination (Fig. 2B). A second dose of either ORFV-S or ORFV-S/N increased the antigen-specific binding antibody titers at day 28 by 1.9- and 3.4-fold, respectively. The IgG2a/IgG1 isotype ratio of RBD-binding antibodies at day 28 showed a TH1-biased profile skewed towards IgG2a for both ORFV-S and ORFV-S/N (Fig. 2C). Virus neutralizing titers (VNT) were similar for both constructs, with a GMT of 2.0×103 and 1.7×103 for ORFV-S and ORFV-S/N, respectively, compared to 4.5 x 102 with the WHO International Standard of anti-SARS-CoV-2 immunoglobulin NIBSC 20/136 (Fig. 2D).
The ORFV-S/N also induced a robust antibody response to the nucleocapsid, which was significantly boosted after the second vaccination (Fig. 1E). The specific IgG2a/IgG1 ratios confirmed a TH1 profile after ORFV-S/N immunization (Fig. 1F).
In addition to the humoral immune response, both ORFV-S and ORFV-S/N recombinants elicited multifunctional CD4 + and CD8 + T cells specific for spike (Fig. 1G, S1A, S1C) and nucleocapsid (Fig. 1H, S1A, S1B), as monitored in spleens seven days after the second vaccination.
The long-term persistence of the vaccine-induced humoral responses as well as boost capacity of the ORFV-based SARS-CoV-2 vaccine candidates was further assessed in mice (Fig. S2A). ORFV-S and ORFV-S/N recombinants elicited elevated RBD-binding total IgG levels over the prolonged follow-up period, and a third vaccination (day 171) boosted pre-existing antibody levels by 2.2- and 1.5-fold for ORFV-S and ORFV-S/N, respectively (Fig. S2B). The antibody levels against nucleocapsid were boosted by 44.6-fold following the third vaccination using ORFV-S/N (Fig. S2C). VNT after the second (day 35) and the third vaccination (day 185) for both ORFV-S and ORFV-S/N were similar (Fig. S2D).
Multi-antigenic ORFV-S/N recombinant confers superior protection to vaccinated Syrian hamsters following SARS-CoV-2 challenge as compared to the mono-antigenic ORFV-S
The Syrian hamster SARS-CoV-2 challenge model is a well-established system to study severe COVID-19 with pathology similar to humans35. In this study, hamsters were vaccinated twice at a 4-week interval with either escalating doses (106 – 108 PFU) or a single dose of the highest dose (108 PFU) of the ORFV-S/N recombinant, or two doses (107 PFU) of ORFV-S recombinant or PBS. In addition, a group of hamsters was infected with SARS-CoV-2 (102 TCID50 intranasally) (ancestral strain; Wuhan) at day 0 (SARS-CoV-2-recovered). All animals were challenged with SARS-CoV-2 (102 TCID50) (ancestral strain; Wuhan) at day 56 (Fig. 3A).
After the first and second vaccination, a dose-dependent increase of RBD-binding IgG antibodies was observed in all groups (Fig. 3B). A correlation was also observed between total antibody levels and VNT.
Four days after SARS-CoV-2 challenge, virus titers were assessed in the nose and lungs, along with lung histopathology. Hamsters vaccinated with ORFV-S and ORFV-S/N and those recovered from a previous SARS-CoV-2 infection showed reduced virus titers in the lungs (Fig. 3D). However, only ORFV-S/N vaccine consistently prevented the presence of virus in the upper respiratory tracts, despite lower antibody and VNT (Fig. 3E).
Histopathological evaluations showed that the ORFV-S/N vaccine conferred protection with minimal affection of the upper and lower airways (Fig. 3F). Vaccination with ORFV-S/N doses at 107 and 108 PFU resulted in the lowest scores of rhinitis, tracheitis, bronchiolitis and alveolitis, whereas two doses of 106 PFU or a single dose of 108 PFU of ORFV-S/N already reduced severity of rhinitis and tracheitis. Interestingly, the ORFV-S recombinant did not protect the vaccinated hamsters from severe rhinitis with scores comparable to the PBS group. However, the histopathology scores of tracheitis and alveolitis were clearly reduced when compared to the PBS group (Fig. 3F). SARS-CoV-2-recovered animals showed increased scores of rhinitis, tracheitis and alveolitis as compared to animals vaccinated with ORFV-S/N, although the scores were lower than those found in the PBS group.
Correlates of protection analyses showed that high VNT elicited by the ORFV-S recombinant did not cluster with reduced viral titers in nasal turbinates, in contrast to the effects observed after one or two administrations of ORFV-S/N. The latter elicited better protection against viral challenge that included the upper airways (Fig. 3G). In addition, no correlation was observed between the VNT after vaccination with both ORFV-S and ORFV-S/N recombinants and the elevated virus titers in lungs after the challenge (Fig. 3H). Clustering of higher VNT levels with reduced histopathology scores suggested possible vaccine dose-dependent effects (Fig. 3I).
The potential of both ORFV-S and ORFV-S/N vaccine candidates to boost pre-existing memory responses after SARS-CoV-2 infection was also investigated in the hamster model. Animals were infected with SARS-CoV-2 (102 TCID50 intranasally) (SARS-CoV-2-recovered) and 42 days later either vaccinated with 3x107 PFU of ORFV-S and ORFV-S/N, or re-infected with SARS-CoV-2. Hamsters were monitored until day 56 (Fig. S3A). Already seven days after the boost with either ORFV-S or ORFV-S/N (day 49), increased RBD-binding IgG titers were observed (Fig. S3B; Table S1). Administration of ORFV-S or ORFV-S/N resulted in 4.5- or 2.6-fold higher RBD-specific antibody levels when compared to re-infection, respectively. Additionally, both vaccinations with the ORFV-S/N recombinant as well as the SARS-CoV-2 re-challenge boosted nucleocapsid-specific antibody levels (Fig. S3C; Table S2). Finally, administration of ORFV-S or ORFV-S/N after SARS-CoV-2 infection increased VNT by 3.6- and 3.5-fold as compared to re-infection, respectively (Fig. S3D; Table S3).
Multi-antigenic ORFV-S/N recombinant protects vaccinated non-human primates from SARS-CoV-2 infection
The superior protection observed in the Syrian hamster model following SARS-CoV-2 challenge prompted an evaluation of ORFV-S/N in a NHP challenge model. NHPs received either two doses of 108 PFU of the ORFV-S/N recombinant with a 4-week interval or PBS as a control (Fig. 4A). Serum and peripheral blood mononuclear cells (PBMCs) were sampled regularly, and animals were challenged with 105 TCID50 SARS-CoV-2, administered into nose and trachea on day 70. Subsequently, SARS-CoV-2 subgenomic messenger RNA (CoVsg) levels were determined daily in nose and throat over one week, and viral titers in bronchoalveolar lavage (BAL) were assessed on day 3 following challenge.
RBD-binding IgG became detectable within 14 days after the first vaccination reaching GMT of 5.0×104 (Fig. 4B). The second vaccination boosted antibody titers by a 9-fold 2 weeks post vaccination. Levels of spike-trimer-binding total IgG and VNT exhibited similar response patterns (Fig. 4C, D). Before challenge, RBD-binding IgG, spike-trimer-binding IgG and neutralizing GMT accounted for 1.4×104, 5.0×104 and 3.4×102, respectively. These values remained at baseline in the PBS group. Antibody levels against nucleocapsid protein stayed at baseline. Both, nucleocapsid- and spike-specific T cell responses with a mean of 69 and 541 IFN-γ spot forming units, respectively, were measured in PBMCs at day 35 by ELISpot (Fig. 4E).
After challenge with 105 TCID50 SARS-CoV-2, half of the vaccinated animals showed detectable viral titers in nose and throat (3/6), and all cleared the virus already at day 4 in contrast to the PBS control group that remained viremic for at least one week (Fig. 4F, G). Viral loads in CoVsg PCR-positive primates were 10- to 100-times lower than in the macaques that received PBS (control). None of the vaccinated NHP showed presence of viral titers in BAL, in contrast to all animals in the control group (Fig. 4H).
To evaluate the duration of ORFV-mediated vaccine protection, NHP were first vaccinated with the ORFV-S/N at a dose of 106 or 3x107 PFU or PBS as a control with a 4-week interval, and then challenged with 105 TCID50 of SARS-CoV-2 seventeen weeks after the last vaccination (Fig. S4). In this long-term experiment, viral loads in CoVsg PCR-positive primates were 2- to 150-times lower than in the macaques that had received PBS (control). In addition, 80% of NHP immunized with the 3x107 PFU of ORFV-S/N recombinant still showed protection against SARS-CoV-2 in the lungs.