SARS-CoV-2 B.1.1.7 infection of Syrian hamster does not cause more severe disease and is protected by naturally acquired immunity

Epidemiological studies have revealed the emergence of multiple SARS-CoV-2 variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality (1). To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and re-infection studies in naïve and convalescent Syrian hamsters (>10 months old). Hamsters infected by either a B.1.1.7 variant or a B.1 (G614) variant exhibited comparable viral loads and pathology. Convalescent hamsters that were previously infected by the original D614 variant were protected from disease following B.1.1.7 challenge with no observable clinical signs or lung pathology. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in hamsters and that natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant.


Introduction
Despite the proofreading activity provided by the 3'-5' exonuclease activity of non-structural protein 14, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accumulated multiple mutations in its viral genome (2). Mutations occurring in the spike protein are of major concern due to the role of this glycoprotein in mediating virus entry and as the major target of neutralizing antibodies (3-6). In March of 2020, the D614G SARS-CoV-2 B.1 variant emerged and became the predominant strain of virus throughout Europe and the United States (7). Since late fall 2020, the emergent B.1.1.7 SARS-CoV-2 variant became predominant in the United Kingdom and contributed to the December surge in positive SARS-CoV-2 cases and the increase in hospitalization and death rates (8). Among the 8 mutations found in the spike protein of the B.1.1.7 variant, the N501Y mutation has been found to increase the binding affinity of spike protein to the human ACE2 receptor, resulting in an elevated transmissibility and monoclonal antibody resistance (9-11).
Here, we set out to address whether a prior exposure to an ancestral SARS-CoV-2 (D614) isolate would offer protection against re-infection by a B.1.1.7 variant. In addition, we investigated whether the B.1.1.7 variant displays fitness advantage over the G614 variant in the Syrian hamster model.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint 4 convalescent hamsters ranged from 80 to 320 at the time of challenge. Nasal wash (NW) samples taken from infected hamsters on days 1, 2 and 3 post infection (PI) showed that all hamsters contained detectable sub-genomic RNA (sgRNA) levels in the nasal cavities on the first two days PI (Fig.1A). Three days following infection, viral sgRNA levels from nasal washes of convalescent hamsters declined to below the detection limit, indicating that convalescent hamsters quickly controlled B.1.1.7 replication. By contrast, naïve hamsters that were challenged with either the B.1.1.7 or B.1. variant showed 2-3 log 10 higher levels of sgRNA compared to the convalescent group. Furthermore, naïve hamsters that were challenged with the B.1.1.7 variant displayed overall higher levels of sgRNA in NW samples (statistically significant at the day 2 PI) than those of the NY B.1 infected group (p=0.0008, 0.01 respectively). The same NW samples were also quantified for infectivity by an 50% tissue culture infectious dose (TCID 50 ) endpoint dilution assay (Fig. 1B). Only 3 out of 12 NW samples from convalescent hamsters exhibited detectable levels of infectious virus at the day 1 PI, dropping to below detection at the day 2 PI (Fig. 1B). Surprisingly, NW samples from NY B.1 infected hamsters contained significantly higher TCID 50 values than those from CA B.1.1.7 infected hamsters on days 2 and 3 PI (p=0.0109, 0.0016 respectively, Fig. 1B).
Convalescent hamsters experienced no weight loss compared to the naïve infected groups over the course of seven days PI (Fig.1C). Both the CA B.1.1.7 and NY B.1 infected animals lost 10-15% of body weight by day 7 PI. At day 7 PI, infected hamster lungs displayed pathology including alveolar wall thickening, airway infiltrates, perivascular edema and hyperplasia ( Fig. 1D-E). However, convalescent hamsters who were subsequently re-infected did not show these signature pathologies in the lungs (Fig. 1E). The cumulative pathology score and percentage of lung consolidation in the CA B.1.1.7 and NY B.1 naïve groups had no significant difference (Fig.   1D), but both had significantly higher scores than those convalescent hamsters (Fig. 1D).
Therefore, although B.1.1.7 SARS-CoV-2 variant replication was higher in the nasal cavity on and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint days 1 and 2 PI compared to the NY B.1 variant measured by sgRNA, the associated pathology was not more severe in the lung.
To account for the discrepancy between sgRNA titers and TCID 50 titers in NW samples, a second study using ten male Syrian hamsters was subsequently performed. This study included two

Discussion
Altogether, this study finds that 1) convalescent hamsters are protected against interstitial pneumonia following B.1.1.7 variant challenge and that the B.1.1.7 variant of SARS-CoV-2 virus does not appear to be more pathogenic in adult male Syrian hamsters when compared to the B.1 variant (D614G). Differences in viral fitness may exist between the B.1.1.7 and B.1 variants in human beings, however, this was not observed in Syrian hamsters which may not be the ideal model to assess that possibility. This study did find an increase B.1.1.7 viral replication concomitant with reduced shedding of infectious virus in NW samples. Such a discrepancy of viral replication and infectious particles may be attributed to a disadvantaged B.1.1.7 plaquing effect in Vero E6 cells which has been previously mentioned (12) which would lead to undercounting of and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. infected hamsters). All sequences remain >99% identity with the sequence of the input virus.
Notably, both the sgRNA and infectious titers of NW samples from the B.1.1.7 infected hamsters are higher than those of the NY B.1 infected hamsters at the Day 4 PI. This finding may suggest a prolonged viral shedding of the B.1.1.7, which has been hypothesized to increase the transmissibility of the B.1.1.7 strain in the human population (11, 13). However, this study did not find any increase in pathogenicity of the B.1.1.7 variant in hamsters. The significance of the increased replication of B.1.1.7 variant in the nasal cavity warrants further exploration through transmission studies. and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint 7

RNA isolation and qRT-PCR
Procedures as described previously (14, 15). and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint 8
Plaque assay. Nasal wash samples were 10-fold serially diluted and added to a 6-well plate with Vero E6 cells. After 1 h the mixture was removed and replenished with Tragacanth gum overlay (final concentration 0.3%). Cells were incubated at 37°C and 5% CO 2 for 2 days, then fixed with 4% paraformaldehyde, followed by staining of cells with 0.1% crystal violet in 20% methanol for 5-10 minutes.

Statistical analysis
Standard unpaired T.test was used to calculate statistical significance through GraphPad Prism (8.4.2) software for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

Acknowledgments
We would like to thank the White Oak FDA Animal Program Staff and Veterinarians: John Dennis, Mario Hernandez, Mario Ortega, and Eric Nimako. We thank Ms. Katherine Shea for assistance with scanning pathology slides. The following reagents were deposited by the Centers for

Diseases Control and Prevention and obtained through Biodefense and Emerging Infections
Research Resources Repository, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281; Isolate USA/CA_CDC_5574/2020, NR-54011; Isolate New York-and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint 9 PV08410/2020, NR-53514. The work described in this manuscript was supported by US FDA intramural grant funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint 1 1  and is also made available for use under a CC0 license.

Figure Legends
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.02.438186 doi: bioRxiv preprint