Prolonged Protective Immunity Induced by Mild SARS-CoV-2 Infection of K18-hACE2 Mice

Abstract

Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show that the infection of K18-hACE2 mice induced robust humoral and cellular immunity (systemic and local), which persisted for at least six months. Virus-specific T cells and neutralizing antibody titers decreased over time, yet their levels were sufficient to provide sterile immunity against lethal rechallenge six months post-primary infection. The study substantiates the role of naturally induced immunity against SARS-CoV-2 infection for preventing recurring morbidity.

Keywords: COVID-19; K18-hACE2 mice; SARS-CoV-2; mild infection.

Figure 1

Humoral and cellular-specific immune response after mild SARS-CoV-2 infection. K18-hACE2 mice were infected with SARS-CoV-2 (80 PFU/mouse, i.n.) 24 weeks (blue), 12 weeks (green) or 3 weeks (red) prior to their simultaneous analysis and challenge. Sera, spleen and lung samples were collected at the indicated time points for the evaluation of SARS-CoV-2-specific humoral (B,C) and cellular (D) responses, as describe in Materials and Methods. (A) Schematic representation of experimental design. (B) SARS-CoV-2 RBD-specific IgG antibodies in the serum of the 3 groups prior to the challenge, determined by ELISA. (C) NT₅₀ titers determined by the Plaque Reduction Neutralization test (PRNT). (D) SARS-CoV-2-specific cellular response determined by ELISpot in the lungs (left panel) and spleen (right panel). Each symbol represents one mouse. p-values indicate significant differences (* p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001).

Figure 2

Protective efficacy against SARS-CoV-2 reinfection. K18-hACE2 mice from the 3 groups, as indicated (see Figure 1A, n = 20 for each group), were reinfected i.n. with 3 × 10⁴ PFU SARS-CoV-2. Control group consisted of naïve animals (n = 15). Animals were monitored daily for body weight (A) and survival (B). Five mice from each group were sacrificed at day 2 and 4 post-challenge. SARS-CoV-2 loads in the lungs (C) and brain (D) were determined by a PFU assay. Data points represent the virus load of each animal; the horizontal bar indicates the mean of each group. The dashed line indicates the lower limit of detection. Statistical analysis was performed with the log-rank (Mantel–Cox) test for survival data.