Influence of SARS-CoV-2 inactivation by different chemical reagents on the humoral response evaluated in a murine model

Abstract

Viral inactivation for antibody induction purposes, among other applications, should ensure biosafety, completely avoiding the risk of infectivity, and preserving viral immunogenicity. β-propiolactone (BPL) is one of the most used reagents for viral inactivation, despite its high toxicity and recent difficulties related to importation, experienced in Brazil during the SARS-CoV-2 pandemic. In this context, the main objectives of this work were to test different inactivation procedures for SARS-CoV-2 and to evaluate the induction of neutralizing antibodies in mice immunized with antigenic preparations obtained after viral treatment with formaldehyde (FDE), glutaraldehyde (GDE), peroxide hydrogen (H₂O₂), as well as with viral proteins extract (VPE), in parallel with BPL. Verification of viral inactivation was performed by subsequent incubations of the inactivated virus in Vero cells, followed by cytopathic effect and lysis plaques observation, as well as by quantification of RNA load using reverse transcription-quantitative real time polymerase chain reaction. Once viral inactivation was confirmed, cell culture supernatants were concentrated and purified. In addition, an aliquot inactivated by BPL was also subjected to viral protein extraction (VPE). The different antigens were prepared using a previously developed microemulsion as adjuvant, and were administered in a four-dose immunization protocol. Antibody production was comparatively evaluated by ELISA and Plaque Reduction Neutralization Tests (PRNT). All immunogens evaluated showed some level of IgG anti-SARS-CoV-2 antibodies in the ELISA assay, with the highest levels presented by the group immunized with FDE-inactivated viral antigen. In the PRNT results, except for VPE-antigen, all other immunogens evaluated induced some level of neutralizing anti-SARS-CoV-2 antibodies, and the FDE-antigen stood out again with the most expressive values. Taken together, the present work shows that FDE can be an efficient and affordable alternative to BPL for the production of inactivated SARS-CoV-2 viral antigen.

Keywords: ELISA; Inactivated virus; PRNT(50); RT-qPCR; SARS-CoV-2.

Graphical abstract

Fig. 1

Determination of amplification efficiency for the molecular quantification of SARS-CoV-2 by the comparative Ct method. (A) Amplification curves generated with four log dilutions of a viral RNA sample equivalent to the initial titer of 1 × 10⁷ PFU/mL. (B) Standard curve generated from the linear region of each amplification curve. Efficiency of amplification was determined using the equation: efficiency (E) = 10^(−1/slope)-1, being E = 90.17% and R² = 0.996.

Fig. 2

Typical cytopathic effect and lysis plaques induced by SARS-CoV-2 infection in Vero cells. (A) Cytopathic effect observed under a light microscope (100X magnification) in Vero E6 cells infected with SARS-CoV-2, (B) compared to non-infected cells, and (C) cell lysis plaques observed after incubation of different viral dilutions (10⁻¹ to 10⁻⁵) in Vero CCL-81 cells, compared to non-infected control cells (CC) in 12-well plate assay.

Fig. 3

Quantification of viral RNA in supernatant from Vero E6 cells challenged with inactivated viral suspensions. The supernatants were collected on days three and seven post-challenge. Gray columns indicate virus inactivated by β-propiolactone (BPL), formaldehyde (FDE), glutaraldehyde (GDE) and hydrogen peroxide (H₂O₂), and black columns represent untreated viral controls. Different symbols indicate significant difference (P > 0.05).

Fig. 4

10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis of SARS-CoV-2 inactivated with formaldehyde (FDE), β-propiolactone (BPL), hydrogen peroxide (H₂O₂), glutaraldehyde (GDE) and virus proteins extracted (VPE). The protein bands were stained with coomassie blue dye.

Fig. 5

Body weight of mice inoculated with different preparations of SARS-CoV-2 antigens. Mean body weight monitored over the tests performed with groups of mice inoculated with antigenic preparations of SARS-CoV-2 inactivated with β-propiolactone (BPL), glutaraldehyde (GDE), formaldehyde (FDE), hydrogen peroxide (H₂O₂), virus proteins extracted (VPE), and control inoculated with non-infected cell supernatant (Mock). No significant difference was observed in any of the experimental groups evaluated (P > 0.05).

Fig. 6

IgG levels detected by ELISA in the plasma of mice immunized with SARS-CoV-2 antigens. The mice groups received a schedule of four immunizations with SARS-CoV-2 inactivated with β-propiolactone (BPL), glutaraldehyde (GDE), formaldehyde (FDE), hydrogen peroxide (H₂O₂), viral protein extract (VPE) and non-infected cell culture supernatant (Mock). Different symbols indicate significant difference (P > 0.05).

Fig. 7

Plaque reduction neutralization test (PRNT) performed with plasma from mice immunized with SARS-CoV-2 antigens. Mice groups received a schedule of four immunizations with SARS-CoV-2 inactivated with β-propiolactone (BPL), formaldehyde (FDE), glutaraldehyde (GDE), hydrogen peroxide (H₂O₂), viral protein extract (VPE) and non-infected cell culture supernatant (Mock). Data are expressed as reciprocal of dilution equivalent to PRNT₅₀. In each column, the open circles correspond to the PRNT₅₀ observed for the animals tested individually, while the closed circles correspond to the PRNT₅₀ observed with the pool of the four animals of each group. Different symbols indicate significant difference (P > 0.05).