Critical role of neutralizing antibody for SARS-CoV-2 reinfection and transmission

Abstract

Cases of laboratory-confirmed SARS-CoV-2 reinfection have been reported in a number of countries. Further, the level of natural immunity induced by SARS-CoV-2 infection is not fully clear, nor is it clear if a primary infection is protective against reinfection. To investigate the potential association between serum antibody titres and reinfection of SARS-CoV-2, ferrets with different levels of NAb titres after primary SARS-CoV-2 infection were subjected to reinfection with a heterologous SARS-CoV-2 strain. All heterologous SARS-CoV-2 reinfected ferrets showed active virus replication in the upper respiratory and gastro-intestinal tracts. However, the high NAb titre group showed attenuated viral replication and rapid viral clearance. In addition, direct-contact transmission was observed only from reinfected ferrets with low NAb titres (<20), and not from other groups. Further, lung histopathology demonstrated the presence of limited inflammatory regions in the high NAb titre groups compared with control and low NAb groups. This study demonstrates a close correlation between a low NAb titre and SARS-CoV-2 reinfection in a recovered ferret reinfection model.

Keywords: COVID-19; SARS-CoV-2; ferret model; neutralizing antibody; reinfection.

Figure 1.

Schedule of SARS-CoV-2 pre- and reinfection in ferrets. To induce varied immune responses, three different doses of SARS-CoV-2 were used to infect groups of ferrets. Following three weeks of infection, serum NAb titres were measured in Vero cells, and ferrets were then grouped according to their NAb titres (NAb < 20, 20–40, 80, and 160) (A). Asterisks indicate statistical significance between each infection group as determined by two-way ANOVA Tukey’s multiple comparisons test (* indicates p < 0.05, ** indicates p < 0.0001). Each group of NMC-nCoV02 (S clade)-primed ferrets and the naive control group were inoculated intranasally with 10^5.0 TCID₅₀ of CBNU-nCoV02 (GH clade) followed by virus and blood collection on day 14 post-infection (B).

Figure 2.

Nasal wash virus titres of ferrets. Virus titres were measured in nasal washes of CBNU-nCoV02-infected ferrets and direct contact sentinel ferrets at 2, 4, 6, 8, 10 days of reinfection. Viral loads in nasal washes were measured by TCID₅₀ (A). The virus RNA copy numbers were measured with qRT-PCR (B). The limit of viral RNA detection with qRT-PCR is 0.3 log₁₀ copies/mL copies per reaction. Data is presented as mean ± SEM. Asterisks indicate statistical significance between the control (Group 1) and each infected group as determined by two-way ANOVA and subsequent Dunnett’s multiple comparisons test (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001).

Figure 3.

Histopathology of lungs following reinfection. Previously infected ferrets were inoculated with 10^5.0 TCID₅₀ of CBNU-nCoV02 (GH clade) virus. Tissues were harvested on day 6 after inoculation. Group 1 (control group) (A), Group 2 (NAb titre < 20) (B), Group 3 (NAb 20–40) (C), Group 4 (NAb titre 80) (D), and Group 5 (NAb titre 160) (E). Magnification 40X.

Figure 4.

Comparison of total IgG and NAb titres between primary infection and reinfection of ferrets. Immunofluorescence assay (IFA) was performed with sera of SARS-CoV-2 infected ferrets using fluorescein-labeled anti-ferret IgG antibody (A and C). Vero cells were infected with 1 × 10³ TCID₅₀/mL NMC-2019-nCoV02 (S clade) (A), CBNU-nCoV02 (GH clade) (C) and incubated with serially diluted ferret sera. Fluorescein-labeled anti-ferret IgG was used as the secondary antibody. NAb titre against NMC-2019-nCoV02 (100 TCID₅₀) (B) and CBNU-nCoV02 (100 TCID₅₀) (D) were measured using Vero cells (B and D). Data are presented as geometric mean ± SD. Asterisks indicate statistical significance compared with primary infection sera by two-way ANOVA Sidak’s multiple comparisons test (* indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.0001).