Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in golden Syrian hamster

Abstract

Reinfections with SARS-CoV-2 have already been documented in humans, although its real incidence is currently unknown. Besides having a great impact on public health, this phenomenon raises the question of immunity generated by a single infection is sufficient to provide sterilizing/protective immunity to a subsequent SARS-CoV-2 re-exposure. The Golden Syrian hamster is a manageable animal model to explore immunological mechanisms able to counteract COVID-19, as it recapitulates pathological aspects of mild to moderately affected patients. Here, we report that SARS-CoV-2-inoculated hamsters resolve infection in the upper and lower respiratory tracts within seven days upon inoculation with the Cat01 (G614) SARS-CoV-2 isolate. Three weeks after the primary challenge, and despite high titres of neutralizing antibodies, half of the animals were susceptible to reinfection by both identical (Cat01, G614) and variant (WA/1, D614) SARS-CoV-2 isolates. However, upon re-inoculation, only nasal tissues were transiently infected with much lower viral replication than those observed after the first inoculation. These data indicate that a primary SARS-CoV-2 infection is not sufficient to elicit a sterilizing immunity in hamster models but protects against lung disease.

Keywords: SARS-CoV-2; animal model; golden Syrian hamster; infection; protection; re-infection; viral variants.

Figure 1.

Experimental design. Golden Syrian hamsters (n = 24, 12 male and 12 female) were intranasally (IN) inoculated with 10^5.8 TCID50 of SARS-CoV-2 Cat01 isolate. Before the challenge blood samples and oropharyngeal swabs (OS) were collected from all animals. At 2-, 4- and 7-days post-inoculation (dpi), 4 infected animals (2 male and 2 female) were euthanized. Before necropsy, blood samples and OS were collected from each animal. Nasal turbinate, lungs and trachea were collected for pathological and virological analyses. In purple, samples used for ELISA and seroneutralization test; in green, samples used for histopathology and immunohistochemistry; in yellow, samples used for RT-qPCR and viral titration in Vero E6 cell. At 21 dpi, the remaining animals (n = 12) were equally divided into two experimental groups. One group was intranasally inoculated with 10^5.2 TCID50 of Cat01 isolate while the other was IN inoculated with WA/1 strain at the same concentration. At day 23-dpi (2 days-post re-inoculation) and 25-dpi (4 days-post re-inoculation), 3 animals/experimental group were euthanized. Sampling was equivalent to that indicated previously. Created with BioRender.com

Figure 2.

Weight variation upon first inoculation and re-challenge. Data are expressed as a percentage of variation referred to the weight recorded at the day of the challenge (a) or re-challenge (b). (a) Mean percentage of weight variation of animals inoculated with SARS-CoV-2 Cat01 variant (blue) or with PBS mock solution (grey). P-value for statistically significant point: 2dpi p < .0001; 3dpi p = .0001; 4dpi p = .0002; 5 dpi p = .0003; 6dpi p < 0.0001; 7dpi p = .0005; 10dpi p = 0.0244. (b) Mean percentage of weight variation of animals after SARS-CoV-2 re-inoculation. In blue animals exposed to SARS-CoV-2 Cat01 variant, in red animals exposed to SARS-CoV-2 WA/1 variant, and in grey animals exposed to PBS mock.

Figure 3.

Pathological findings in lungs of hamsters after inoculation and re-inoculation. (a–e) Histopathological findings in lungs of hamsters after SARS-CoV-2 Cat01 challenge on 2 (a), 4 (b) and 7 (c) dpi, and 2 (d) and 4 (e) dpri with Cat01 and WA/1 variants. Broncho-interstitial pneumonia (asterisks) severity increased from 2 to 7 dpi (maximum lesion severity) and was residual at 2 and 4 dpri. Inset in 2a displays submucosa mononuclear inflammation of the bronchus (asterisk) and exocytosis through the epithelium. Hematoxylin and eosin stain, 100× magnification (inset in 2a, 400× magnification) (f to j). Immunohistochemical findings in lungs of same animals. High amount of viral antigen (brown staining) mainly in bronchi epithelium as well moderate amount at 2 dpi (f, inset shows a detail of the bronchus epithelial labelling). The maximum amount of labelling in lung parenchyma, associated with the inflammatory infiltrate, was detected at 4 dpi (g). Scarce number of stained cells were detected at 7 dpi (h, arrowhead) and no labelling was recorded at 2 (i) and 4 (j) dpri. Immunohistochemistry to detect the NP of SARS-CoV-2 and hematoxylin counterstain, 100× magnification (inset in 2a, 400× magnification). Scale bars, 100 μm.

Figure 4.

Viral loads in samples obtained from hamsters after inoculation and re-inoculation with SARS-CoV-2. Genomic RNA (a) and subgenomic RNA levels (b) of SARS-CoV-2 was analyzed in oropharyngeal swabs (OS), nasal turbinate, trachea, and lungs, as well as the infectious viral loads (c). Horizontal bars reflect median viral loads. In blue data obtained from Cat01-reinfected animals, in red data obtained from WA/1-reinfected animals. Dotted lines indicate the limit of detection for each technique. Statistically, significant p values are reported in the graph, preceded by an (A) for ANOVA test or (K) for Kruskal–Wallis tests.

Figure 5.

Humoral responses in SARS-CoV-2 reinfected hamster. (i) Antibody subclasses against (a) Spike protein subunits 1 and 2, (b) receptor-binding domain (RBD) and (c) nucleocapsid protein. In black, serum samples from animals challenged with Cat01 (1st inoculum), in blue serum from animals re-inoculated with the same viral variant (Cat01), in red serum from animal re-inoculated with the different viral variant (WA/1). At 21 dpi before the re-inoculation sera were collected from Cat01-exposed animals, pooled following housing criteria and analyzed (in gold). In grey, serum from control animals treated with a mock solution of PBS. (ii) Serum from all animals was used for live virus neutralization assay against d) Cat01 and e) WA/1 variants. Code colour are the same than those used in panel (i). Pooled sera were not tested in the SNT assay. Dotted lines indicate the limit of detection of the technique.