Toxicity and Local Tolerance of a Novel Spike Protein RBD Vaccine Against SARS-CoV-2, Produced Using the C1 Thermothelomyces Heterothallica Protein Expression Platform

Abstract

Coronavirus disease 2019 (COVID-19) has caused the ongoing COVID-19 pandemic and there is a growing demand for safe and effective vaccines. The thermophilic Thermothelomyces heterothallica filamentous fungal host, C1-cell, can be utilized as an expression platform for the rapid production of large quantities of antigens for developing vaccines. The aim of this study was to evaluate the local tolerance and the systemic toxicity of a C1-cell expressed receptor-binding domain (C1-RBD) vaccine, following repeated weekly intramuscular injections (total of 4 administrations), in New Zealand White rabbits. The animals were sacrificed either 3 days or 3 weeks following the last dose. No signs of toxicity were observed, including no injection site reactions. ELISA studies revealed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G antibodies in the sera of C1-RBD-treated animals starting from day 13 post injection, that were further elevated. Histopathology evaluation and immunohistochemical staining revealed follicular hyperplasia, consisting of B-cell type, in the spleen and inguinal lymph nodes of the treated animals that were sustained throughout the recovery phase. No local or systemic toxicity was observed. In conclusion, the SARS-CoV-2 C1-RBD vaccine candidate demonstrated an excellent safety profile and a lasting immunogenic response against receptor-binding domain, thus supporting its further development for use in humans.

Keywords: C1; COVID-19; RBD; SARS-CoV-2; Thermothelomyces heterothallica; rabbits; safety; toxicity; vaccine.

Figure 1.

(A-C) Injection site (skeletal muscle) from an animal injected with the placebo (group 1, control item), sacrificed 24 days post first dosing. The lesions consist of mild (grade 2) interstitial infiltration of histiocytes (foreign body granulomatous reaction) associated with degeneration/necrosis of these cells (arrow). No necrosis of the adjacent myofibers was noted. Figure A—original objective x 4; Figure B—original objective x 20; Figure C—original objective x 40. (D-F) Injection site (skeletal muscle) from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 24 days post first dosing. In addition to the mild interstitial infiltration of histiocytes (foreign body granulomatous reaction) associated with degeneration/necrosis of these cells, mild interstitial infiltration of mixed polymorphonuclear cells (heterophils) and mononuclear cells (arrowheads) were noted. No necrosis of the adjacent myofibers was noted. Figure D—original objective x 4; Figure E—original objective x 20; Figure F—original objective x 40. (G, H) The iliac lymph node from an animal injected with the placebo (group 1, control item), sacrificed 24 days post first dosing. Note, no evidence of germinal centers (normal appearance, compared with Figures J and K). Figure G—original objective x 10; Figure H—original objective x 20. (I) The iliac lymph node from an animal injected with the placebo (group 1, control item), sacrificed 24 days post first dosing. Note, no evidence of germinal centers (compared with Figure L). Staining with antibody for PAX-5 (Biocare’s PAX clone BC/24, 1:100). (J, K) The iliac lymph node from animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 24 days post first dosing. The lesions consist of mild germinal centers with increased lymphocytic cellularity (ie, follicular hyperplasia; compared with Figures G and H). Figure J— original objective x 10; Figure K—original objective x 20. (L) The iliac lymph node from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 24 days post first dosing. The lesions consist of mild germinal centers with increased lymphocytic cellularity (ie, follicular hyperplasia). Note the positive staining of the germinal centers (arrows; compared with Figure I). Staining with antibody for PAX-5 (Biocare’s PAX clone BC/24, 1:100). (M, N) The spleen from an animal injected with the placebo (group 1, control item), sacrificed 24 days post first dosing. Note, no evidence of germinal centers (normal appearance, compared with Figures P and Q). Figure M—original objective x 10; Figure N—original objective x 20. (O) The spleen from an animal injected with the placebo (group 1, control item), sacrificed 24 days post first dosing. Note, no evidence of germinal centers (compared with Figure R). Staining with antibody for PAX-5 (Biocare’s PAX clone BC/24, 1:100). (P, Q) The spleen from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 24 days post first dosing. The lesions consist of mild germinal centers with increased lymphocytic cellularity (ie, follicular hyperplasia; compared with Figures M and N). Figure P—original objective x 10; Figure Q— original objective x 20. (R) The spleen from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 24 days post first dosing. The lesions consist of mild germinal centers with increased lymphocytic cellularity (ie, follicular hyperplasia). Note the positive staining of the germinal centers (arrows; compared with Figure O). Staining with antibody for PAX-5 (Biocare’s PAX clone BC/24, 1:100). (S, T) The injection site (skeletal muscle) from an animal injected with the placebo (group 1, control item), sacrificed 42 days post first dosing (recovery phase). The lesions consist of mild (grade 2) interstitial infiltration of histiocytes (foreign body granulomatous reaction) associated with degeneration/necrosis of these cells (arrows). No necrosis of the adjacent myofibers was noted. Figure S—original objective x 4; Figure T—original objective x 20. (U, V). The injection site (skeletal muscle) from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 42 days post first dosing (recovery phase). The lesions consist, in addition to the mild interstitial infiltration of histiocytic (foreign body granulomatous reaction) associated with degeneration/necrosis of these cells (arrows), also of minimal interstitial infiltration of mixed polymorphonuclear cells (heterophils) and mononuclear cells (arrowheads). No necrosis of the adjacent myofibers was noted (arrows). Figure U—original objective x 4; Figure V—original objective x 20. (W) The iliac lymph node from an animal injected with the placebo (group 1, control item), sacrificed 42 days post first dosing (recovery phase). Note, no evidence of germinal centers (normal appearance, compared with Figure Z). Arrowheads indicate histiocytic cell infiltration. Figure W—x 20. (X) The iliac lymph node from an animal injected with the placebo (group 1, control item), sacrificed 42 days post first dosing. Note, no evidence of germinal centers (compared with Figure Z). Staining with antibody for PAX-5 (Biocare’s PAX clone BC/24, 1:100). (Y) The iliac lymph node from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 42 days post first dosing (recovery phase). The lesions consist of mild germinal centers with increased lymphocytic cellularity (ie, follicular hyperplasia). Arrowheads indicate histiocytic cell infiltration (compared with Figure W). Figure Y—original objective x 20. (Z) The iliac lymph node from an animal injected with the C1 SARS-CoV-2 RBD vaccine candidate (group 2, test item), sacrificed 42 days post first dosing. The lesions consist of mild germinal centers with increased lymphocytic cellularity (ie, follicular hyperplasia). Note the positive staining of the germinal centers (arrows; compared with Figure X). Staining with antibody for PAX-5 (Biocare’s PAX clone BC/24, 1:100).