Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration

Abstract

Introduction: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease.

Materials and methods: We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms.

Results: Using HLA-A^*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A^*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies.

Conclusion: PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.

Keywords: T cell epitopes; T-cell-directed vaccine; coronaviruses; dwarf hamster COVID-19 model; multi-epitope vaccine; pan-coronavirus vaccine; universal COVID-19 vaccine.

Figure 1

PanCoVac design. Structural proteins (S, E, M, N) from coronaviruses were analyzed. Conserved immunogenic peptides from all structural proteins were identified by bioinformatics analyses. The conserved immunogenic peptides from each protein were linked together with double alanine (AA) spacers resulting in four polypeptide blocks which were fused together by furin cleavage sites. An IgE leader sequence was attached to the N terminus. In order to investigate processing and presentation, the HPV 16 E6₂₉₋₃₈ (TIHDIILECV) epitope, which binds to HLA-A ^* 0201, was inserted in the middle of the S module (PanCoVacE6). The amino acid sequences of PanCoVac and PanCovacE6 were codon optimized for human expression and cloned into pLeGo-iG2 plasmids (pLeGo-iG2-PanCoVac, pLeGo-iG2-PanCoVacE6).

Figure 2

Stimulation of HPV E6-specific reporter T cells by PanCoVacE6 expressing cells. (A) U251 cells were transduced with PanCoVac or PanCoVacE6 using LV or NILV. In addition, U251 cells were transfected with either EGFP mRNA or PanCoVacE6 mRNA. After 18 h, the medium was removed and HPV E6 peptide-specific reporter cells were added at a ratio 2:1 for 24 h. Subsequently, the cells were collected, washed and stained with BV711 mouse anti-human CD3 antibody and live/dead Zombie Violet dye. Stimulation of reporter cells is given as percentage of maximal peptide stimulation, i.e. stimulation of reporter cells with U251 cells pulsed with HPV E6 peptide (TIHDIILECV). Results are derived from at least 5 independent experiments; error bars represent the mean ± SEM.; ***P < 0.001; **P < 0.01; ns not significant, unpaired t-test. (B) U251 cells were transfected with PanCoVacE6Δfurin mRNA (control) and PanCoVacE6 mRNA, respectively. HPV E6 peptide-specific reporter cells were added to transfected U251 cells as described in (A). The results are shown as fold change relative to the control. Results are derived from 4 independent experiments; error bars represent the mean ± SEM.; *P < 0.05, unpaired t-test.

Figure 3

Body weight, body temperature and lung histopathology after challenge of vaccinated dwarf hamsters with SARS-CoV-2. Body temperature (A) and body weight (B) of dwarf hamsters vaccinated with NILV-PanCoVac or NILV were monitored on a daily basis until the experiment was terminated at 7 days post infection (dpi).

Figure 4

Representative histopathology of lung tissue derived from NILV or NILV-PanCoVac vaccinated Roborovski dwarf hamsters after challenge with SARS-CoV-2. At 2 dpi, bronchioli with mild bronchiolitis and moderate epithelial cell necrosis (arrow heads; left panel) were observed. The respiratory parenchyma presented with moderate to severe inflammation, alveolar wall necrosis (circle; central panel) and alveolar edema (asterisk; central panel). Histopathological analysis of blood vessels revealed endothelialitis (arrows; right panel). Haematoxylin and eosin stain; bars represent 20 µm.

Figure 5

Histopathological scores of lung tissue derived from NILV or NILV-PanCoVac vaccinated Roborovski dwarf hamsters after challenge with SARS-CoV-2. At 2 days post infection (dpi), 5 dpi and 7 dpi, three animals of each group were sacrificed and histopathological changes in the lung were scored using a four-scale severity grading system (0: no lesions, 1: mild, 2: moderate, and 3: severe). The cumulative results are also shown.

Figure 6

Titers of SARS-CoV-2 neutralizing antibodies in sera and SARS-CoV-2 quantification in oropharyngeal swabs derived from of infected dwarf hamsters. Roborovski dwarf hamsters (P. roborovskii) were immunized i.n. with 1×10⁵ particles either NILV-PanCoVac (9 animals) or NILV (9 animals). After 21 days, the hamsters were challenged with a sublethal dose (1×10⁴ pfu) of the ancestral SARS-CoV-2 (Wuhan) strain. At 2 dpi, 5 dpi, and 7 dpi, three animals of each group were sacrificed and sera and oropharyngeal swabs were collected for further analysis. (A) Titers of SARS-CoV-2 neutralizing antibodies were determined. Shown is the maximal serum dilution that still completely neutralized SARS-CoV-2 in a cell culture assay. (B) SARS-CoV-2 genome copy numbers in oropharyngeal swabs derived from animals vaccinated either with NILV-PanCoVac or NILV (control) were determined. One animal in the NILV-vaccinated group scheduled for the analysis at 7 dpi (Hamster No. 8) died at 4 dpi. The corresponding serum and oropharyngeal swab, respectively was analyzed together with the probes scheduled for 5 dpi resulting in 4 measured values at this time point.

Figure 7

SARS-CoV-2 quantification in lung tissue from Roborovski dwarf hamsters after challenge with SARS-CoV-2. Roborovski dwarf hamsters (P. roborovskii) were immunized i.n. (1×10⁵ particles) with NILV-PanCoVac (9 animals) or NILV (9 animals). After 21 days, the hamsters were challenged with a sublethal dose (1×10⁴ pfu) of the ancestral SARS-CoV-2 (Wuhan) strain. At 2 dpi, 5 dpi and 7 dpi, three animals of each group were sacrificed and lung tissue was analyzed. (A) SARS-CoV-2 genome copy numbers per 2.5 mg lung tissue derived from animals vaccinated either with NILV-PanCoVac or NILV (control) were determined at the indicated time points (left graph). The cumulative results are also shown (right graph). (B) Virus titers in 50 mg lung tissues from animals vaccinated either with NILV-PanCoVac or NILV (control) were analyzed at the indicated time points (left graph). The cumulative results are also shown (right graph). Error bars represent the mean ± SEM; ***P < 0.001; **P < 0.01; *P < 0.05, unpaired t-test. One animal in the control group (Hamster No. 8) scheduled for the analysis at day 7 dpi died at 4 dpi. The corresponding lung tissue was analyzed together with the probes scheduled for 5 dpi resulting in 4 measured values at this time point.