Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B

Abstract

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle–formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.

Fig. 1.. SARS-CoV-2 mRNA vaccine candidates elicit SARS-CoV-2 nAbs in mice.

(A) Schematic representation of the mRNA vaccine constructs. S, SARS-CoV-2 Spike mRNA (amino acids 1 to 1273); S_furinmut, SARS-CoV-2 Spike mRNA (amino acids 1 to 1273) in which the furin cleavage site was removed by replacing NSPRRA (amino acids 679 to 684) with IL; RBD, SARS-CoV-2 RBD (receptor binding domain) mRNA (amino acids 319 to 541); tdTomato, control mRNA encoding tdTomato; S1, S1 subunit of SARS-CoV-2 Spike (amino acids 1 to 685); S2, S2 subunit of SARS-CoV-2 Spike (amino acids 686 to 1273). (B) Mice vaccination regimen. Six- to 8-week-old mice were vaccinated twice with a 3-week interval. One day before each vaccination, peripheral blood was collected from the mice. Three weeks after the second vaccination, mice were humanely euthanized, and blood and spleens were collected from the mice. (C) C57BL/6 mice (n = 8 per group) were vaccinated with 20 μg of mRNA vaccine candidates (S, S_furinmut, and RBD) or control mRNA tdTomato or DPBS for naïve control mice. Three weeks after the second vaccination, blood was collected to test neutralization of SARS-CoV-2 authentic virus or pseudovirus by the sera. For comparison, convalescent sera from eight SARS-CoV-2–infected human subjects (HCS in the graph) were also tested for neutralization of SARS-CoV-2 authentic virus. Each symbol represents one mouse or person. Samples that did not neutralize viruses at the lowest dilution (1:20 for real virus and 1:40 for pseudovirus) are designated an ID₅₀ titer of 1. For each group, the long horizontal line indicates the median, and the short lines below and above the median indicate the 25th and 75th percentiles. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by one-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple comparison test.

Fig. 2.. PTX-COVID19-B elicits potent humoral immune responses in mice.

(A and B) Female C57BL/6 mice were vaccinated with 1 or 10 μg of PTX-COVID19-B or 10 μg of control tdTomato mRNA. Three weeks after the second vaccination, blood was collected to detect (A) S-specific binding antibodies in the mouse sera as measured by ELISA and (B) neutralization of SARS-CoV-2 authentic virus or pseudovirus by the mouse sera. Shown in (A) are EC₅₀ titers. N = 10 for each of the PTX-COVID19-B group. Shown in (B) are ID₅₀ titers. N = 10 per group. (C) Six- to 8-week-old male and female BALB/c mice were vaccinated with 4- or 20-μg doses of PTX-COVID19-B or formulation buffer as a control. Three weeks after the second vaccination, blood was collected to detect serum neutralization of SARS-CoV-2 authentic virus or pseudovirus by the mouse sera. Shown are ID₅₀ titers. N = 10 per group except for n = 9 for the female 20 μg–dosed PTX-COVID19-B group in the pseudovirus assay. In (B) and (C), samples that did not neutralize viruses at the lowest dilution (1:20 for real virus and 1:40 for pseudovirus) are designated an ID₅₀ titer of 1. Each symbol represents one mouse. For each group, the long horizontal line indicates the median, and the short lines below and above the median indicate the 25th and 75th percentiles. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by one-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple comparison test.

Fig. 3.. Neutralization of VOCs by immune sera from PTX-COVID19-B vaccinated mice.

(A) Schematic representation of S proteins from SARS-CoV-2 VOCs, highlighting mutated amino acids compared to the ancestral Wuhan-Hu-1 isolate (triangles denoting deletions and rhombuses denoting replacements). (B to D) Neutralization of pseudoviruses bearing S protein from VOCs and Wuhan-Hu-1 isolate. C57BL/6 mice immune sera shown in Fig. 2 (A and B) were used in the neutralization. Shown in (B) are ID₅₀ titers across all pseudoviruses except VOC Delta. Shown in (C) are pairwise comparisons of the ID₅₀ titers between VOCs and the Wuhan-Hu-1 isolate. Shown in (D) is a pairwise comparison of the ID₅₀ titers between VOC Delta and the Wuhan-Hu-1 isolate. The numbers above the brackets in (C) and (D) are the ratios of the median ID₅₀ titers against the VOCs to the titers against Wuhan-Hu-1 isolate (blue, 10-μg PTX-COVID19-B group; red, 1-μg PTX-COVID19-B group). Each symbol represents one mouse. *P < 0.05 as determined by two-tailed paired t test.

Fig. 4.. PTX-COVID19-B elicits robust cellular immune responses in mice.

(A, C, and D) Female C57BL/6 mice were vaccinated with 1- or 10-μg doses of PTX-COVID19-B or 10 μg of tdTomato mRNA. (B) Male and female BALB/C mice were vaccinated with 4- or 20-μg doses of PTX-COVID19-B or formulation buffer as a control. Three weeks after the second vaccination, spleens were collected and the splenocytes were stimulated with SARS-CoV-2 S peptide pool to detect cytokine production, as measured by ELISPOT shown in (A) and (B), intracellular cytokine staining and flow cytometry shown in (C), and a multiplex immunoassay shown in (D). Shown in (A) are the numbers of IFN-γ spot-forming units (SFU) per million splenocytes (n = 10 per group), (B) the numbers of IFN-γ and IL-4 spot-forming units per million splenocytes (n = 9 for each of the formulation groups and n = 10 for each of the PTX-COVID19-B groups), (C) percentage of cytokine-producing cells in CD4⁺ or CD8⁺ T cells (n = 10 per group), and (D) quantity of the cytokines in the supernatants of the stimulated splenocytes (n = 10 per group). Each symbol represents one mouse. For each group, the long horizontal line indicates the mean, and the short lines below and above the mean indicate the SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by one-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple comparison test.

Fig. 5.. PTX-COVID19-B protects mice from SARS-CoV-2 challenge.

(A) Mouse vaccination and challenge regimen. Six- to 8-week-old female C57BL/6 mice (n = 10 per group) were vaccinated twice with 1-, 4-, or 20-μg doses of PTX-COVID19-B or formulation buffer as a control. One week after the second vaccination, mice were intranasally transduced with AAV6-hACE2. Nine days after the transduction, mice were intranasally challenged with SARS-CoV-2. One day before each vaccination, blood was collected from the mice. Four days after SARS-CoV-2 challenge, mice were humanely euthanized, and blood and lungs were collected from the mice. (B) Amount of infectious SARS-CoV-2 virus and (C) SARS-CoV-2 RNA in the lungs of the mice. Shown in (B) is TCID₅₀/100 mg lung tissue (n = 10 per group) and (C) RNA copies/mg lung tissue (n = 10 per group). (D) Neutralization of SARS-CoV-2 authentic virus by the mouse sera collected 4 days after SARS-CoV-2 challenge. Shown are ID₅₀ titers (n = 10 per group). Samples that did not neutralize viruses at the lowest dilution (1:20) are designated an ID₅₀ titer of 1. Each symbol represents one mouse. For each group, the long horizontal line indicates the median, and the short lines below and above the median indicate the 25th and 75th percentiles. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by one-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple comparison test.

Fig. 6.. PTX-COVID19-B protects hamsters from SARS-CoV-2 challenge.

(A) Hamster vaccination and SARS-CoV-2 challenge regimen. Six- to 10-week-old male Syrian hamsters (n = 8) were vaccinated with a 20-μg dose of PTX-COVID19-B or formulation buffer twice with a 3-week interval. Twenty days after the second vaccination, hamsters were challenged intranasally with SARS-CoV-2. Four and 8 days after the challenge, half of the animals in each group (four animals per group) were humanely euthanized, and blood and tissues were collected. (B) Body weight of the hamsters. Symbols indicate the means, and the error bars indicate the SEM for each group. N = 8 per group from days 0 to 3. N = 4 from days 5 to 8. (C) Amount of infectious SARS-CoV-2 virus in the hamster tissues. Shown are TCID₅₀ values per milliliter of the tissue homogenates (lung and nasal turbinates) or per milliliter of oral swab samples. For lungs and nasal turbinates, each symbol represents one hamster (n = 4 per group per time point), the long horizontal line indicates the median, and the short lines below and above the median indicate the 25th and 75th percentiles. For oral swabs, symbols indicate the medians, and the error bars indicate the 25th and 75th percentiles (n = 8 per group from days 0 to 3 and n = 4 from days 5 to 7). *P < 0.05 and ****P < 0.0001 as determined by two-way ANOVA followed by Sidak’s multiple comparison test.

Fig. 7.. PTX-COVID19-B protects hamster lungs from the pathology caused by SARS-CoV-2 challenge.

Lung tissues obtained from the hamsters in Fig. 6 were fixed in formalin and stained with hematoxylin and eosin. (A) Representative micrographs of the hamster lungs, showing extensive acute and mixed inflammatory cell infiltrates in bronchiole and alveoli in a control hamster receiving formulation buffer and paucity of inflammation in a vaccinated animal after challenged with SARS-CoV-2. Scale bars, 100 μm. (B) Pathology score of the hamster lungs. Shown are semiquantitative pathology scores of the hamster lungs. Each symbol represents one hamster (n = 4 per group per time point). The long horizontal lines indicate the means, and the short lines below and above the mean indicate the SEM. **P < 0.01 and ***P < 0.001 as determined by two-way ANOVA followed by Sidak’s multiple comparison test.