key: cord-0846868-5qvvx566 authors: Brustolin, Marco; Rodon, Jordi; de la Concepción, María Luisa Rodríguez; Ávila-Nieto, Carlos; Cantero, Guillermo; Pérez, Mónica; Te, Nigeer; Noguera-Julián, Marc; Guallar, Víctor; Valencia, Alfonso; Roca, Núria; Izquierdo-Useros, Nuria; Blanco, Julià; Clotet, Bonaventura; Bensaid, Albert; Carrillo, Jorge; Vergara-Alert, Júlia; Segalés, Joaquim title: Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in hamsters date: 2021-01-07 journal: bioRxiv DOI: 10.1101/2021.01.07.425729 sha: 8ebbdac607fb73643d8d6d6778e0e07f20151f63 doc_id: 846868 cord_uid: 5qvvx566 Reinfections with SARS-CoV-2 have already been documented in humans, although its real incidence is currently unknown. Besides having great impact on public health, this phenomenon raises the question if 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 primary challenge, and despite high titers 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. India 8-12 . The symptoms described in these cases had different degrees of severity 65 compared to the first infectious event and ranged from asymptomatic to severe disease, 66 being more intense during the second infections in few patients. In all cases, differences 67 in viral genomic sequences were identified between the first and second infections. 68 4 Experimental reinfection studies have been performed in non-human primates (NHPs), 69 transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2), 70 Cyclophosphamide (CyP) immunosuppressed and RAG2-knockout Golden Syrian 71 hamster (Mesocricetus auratus) and cat (Felis catus) [13] [14] [15] [16] [17] . In all models, animals re-72 challenged with the same SARS-CoV-2 isolate developed a protective non-sterilizing 73 immunity. Currently, there is no data available about the induction of protective 74 immunity conferred by a given strain versus another variant. 75 The Golden Syrian Hamster is a suitable model to study 26 . It is therefore important to gain insights into 97 mechanisms of reinfection and the development of protective immunity using different 98 viral strain, which could interfere with a primary infection event. 99 Our results demonstrate that animals exposed to Cat01 variant developed a cross- Moderate to severe inflammatory lesions were observed in nasal turbinates at 2 and 4 126 dpi, being mild at 7 dpi. Animals developed multifocal to diffuse, muco-purulent to non-127 suppurative rhinitis, which was more evident in the mid and caudal turbinates. Epithelial 128 cell cilia loss was observed multifocally at 2 and 4 dpi. At 7 dpi, the same lesions were 129 observed but considered mild. Upon re-inoculation, nasal turbinates showed mild 130 lesions at 23 dpi, that is 2 days post-re-inoculation (2 dpri), and dpi 25 (4 dpri), like those 131 observed on 7 dpi. The amount of SARS-CoV-2 nucleocapsid protein (NP) detected by 132 immunohistochemistry (IHC) in nasal turbinates correlates with the intensity of lesions, 133 showing high, moderate and low amounts of viral antigen at 2 dpi (Supplementary figure 134 1 (Sp1), a), 4 dpi (Sp1, b) and at 7 dpi (Sp1, c) respectively. Upon re-inoculation, the 135 amount of labelling tended to be mild-to-moderate at 23 dpi (2 dpri) (Sp1, d-e) and very 136 mild at 25 dpi (4 dpri) (Sp1, f-g). Viral antigen was mainly located in nasal epithelial cells, A previous SARS-CoV-2 priming prevents re-infection of the lower respiratory tract 173 We assessed viral genomic and subgenomic SARS-CoV-2 RNA (gRNA and sgRNA, 174 respectively) levels in nasal turbinates, trachea, lungs and oropharyngeal swabs at 2, 4 175 and 7 dpi (n=4/day) and 2 and 4 dpri (n=3/day/viral variant) (Figure 3 ). In addition, we 176 analyzed gRNA and sgRNA levels in oropharyngeal swabs (OS) before the re-challenge 177 to confirm that animals cleared the infection. 178 After the first inoculation, SARS-CoV-2 gRNA loads peaked at 2 dpi in all anatomical 179 compartments and then progressively decreased, accordingly to previous findings 19 . 180 Viral loads in oropharyngeal swabs were similar at 2 and 4 dpi but decreased by 7 dpi Table 2 ). Conversely, we were not able to identify infectious ). Seroconversion against all tested proteins was evident by 7 dpi, although S 232 specific antibody levels were notably lower than those against the RBD and NP, probably 233 due to a lower sensitivity of the S-ELISA compared with the RBD one. Data obtained from 234 sera collected at 21 dpi suggested that total antibody levels targeting the S glycoprotein 235 11 and those recognizing specifically the RBD subdomain incremented between 7 and 21 236 dpi. Conversely, the level of NP-specific antibodies notably decreased at 21 dpi. After re-237 inoculation, the levels of antibodies against S, RBD and NP antigens further increased 238 until 25 dpi (4 dpri) independently of the strain. 239 We then evaluated the neutralization activity of sera obtained from all animals excepted 240 those collected at 21 dpi. Neutralization activity was detected from 7 dpi onwards, and 241 sharply increased at 2 and 4 dpri (Figures 4d and 4e) , similar to kinetic profiles of antigen- WHO. WHO Coronavirus Disease (COVID-19) Dashboard | WHO Coronavirus Disease (COVID-19) Dashboard Human neutralizing antibodies elicited by SARS-CoV-2 infection. 502 Robust neutralizing antibodies to SARS-CoV-2 infection 504 persist for months. 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Science (80-. ) Figure 1. Weight variation upon first inoculation and re-challenge. Data are expressed 604 as percentage of variation referred to the weight recorded at the day of the challenge 605 (a) or re-challenge (b). a) Mean percentage of weight variation of animals inoculated 606 with SARS-CoV-2 Cat01 variant (blue) or with PBS mock solution (grey). b) Mean 607 percentage of weight variation of animals after SARS-CoV-2 re-inoculation. In blue 608 animals exposed to SARS-CoV-2 Cat01 variant WA/1 variant, and in grey animals exposed to PBS mock solution Figure 2. Pathological findings in lungs of hamsters after inoculation and re-613 inoculation 4 (b) and 7 (c) dpi, and 2 (d) and 4 (e) dpri with Cat01 and WA/1 615 variants. Broncho-interstitial pneumonia severity increased from 2 to 7 dpi (maximum 616 lesion severity) and was residual at 2 and 4 dpri Hematoxylin and eosin stain, 100x magnification High amount of viral antigen 620 mainly in bronchi epithelium as well moderate amount at 2 dpi (f, inset shows a detail 621 of the bronchus epithelial labelling). The maximum amount of labelling in lung 622 parenchyma, associated to the inflammatory infiltrate, was detected at 4 dpi (g) Viral loads in samples obtained from hamsters after inoculation and re-629 inoculation with SARS-CoV-2. Genomic RNA (a) and subgenomic RNA levels (b) of SARS-630 Horizontal bars reflect median viral loads. In blue 632 31 data obtained from Cat01-reinfected animals, in red data obtained from WA/1-633 reinfected animals. Statistically significant p values are reported in the graph Humoral responses in SARS-CoV-2 reinfected hamster. i) Antibody subclasses 637 against a) Spike protein subunits 1 and 2, b) receptor binding domain In black, serum samples from animals challenged with Cat01 (1st 639 inoculum), in blue serum from animals re-inoculated with the same viral variant (Cat01) In grey, serum from control animals 643 treated with mock solution of PBS. ii) Serum from all animals were used for live virus 644 neutralization assay against d) Cat01 and e) WA/1 variants. Code color are the same that 645 those used in panel i) Golden Syrian hamsters (n=24, 12 male and 12 female) 649 were intranasally (IN) inoculated with 10 5.8 TCID50 of SARS-CoV-2 Cat01 isolate. Before 650 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 Before necropsy, blood samples and OS were collected from each animal At 21 dpi, the remaining animals (n = 12) 657 were equally divided in two experimental groups. One group was intranasally inoculated 658 with 10 5.2 TCID50 of Cat01 isolate while the other was IN inoculated with WA/1 strain at 659 the same concentration. At day 23-dpi (2 days-post re-inoculation) and 25-dpi (4 days-660 post re-inoculation approach based on the amount of inflammation (none, mild, moderate, or severe) was 426 used to score the damage caused by SARS-CoV-2 infection in hamsters. 427 A previously described immunohistochemistry technique to detect SARS-CoV-2 NP 428 antigen 40 using the rabbit monoclonal antibody (40143-R019, Sino Biological, Beijing, 429 China) at dilution 1:1000, was applied on nasal turbinates, trachea, lung and mediastinal 430 lymph nodes. The amount of viral antigen in tissues was semi-quantitatively scored in 431 the different studied tissues (low, moderate and high amount, or lack of antigen 432 detection).