key: cord-0830811-er4t88lq
authors: Patel, Devanshi R.; Field, Cassandra J.; Septer, Kayla M.; Sim, Derek G.; Jones, Matthew J.; Heinly, Talia A.; McGraw, Elizabeth A.; Sutton, Troy C
title: Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate
date: 2020-11-22
journal: bioRxiv
DOI: 10.1101/2020.11.20.392381
sha: e89aaedfde9e2d7372ebdd70f4c6276b4bda20c5
doc_id: 830811
cord_uid: er4t88lq

SARS-CoV-2 has initiated a global pandemic and vaccines are being rapidly developed. Using the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the ability of prior infection or vaccine-induced immunity to protect against infection in ferrets. Ferrets were semi-permissive to infection with the USA-WA1/2020 isolate. When transmission was assessed via the detection of vRNA at multiple timepoints, direct contact transmission was efficient to 3/3 and 3/4 contact animals in two respective studies, while respiratory transmission was poor to only 1/4 contact animals. To assess the durability of immunity, ferrets were re-challenged 28 or 56 days post-primary infection. Following viral challenge, no infectious virus was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared. To determine if intramuscular vaccination protected ferrets against infection, ferrets were vaccinated using a prime-boost strategy with the S-protein receptor-binding domain formulated with an oil-in-water adjuvant. Upon viral challenge, none of the mock or vaccinated animals were protected against infection, and there were no significant differences in vRNA or infectious virus titers in the nasal wash. Combined these studies demonstrate that in ferrets direct contact is the predominant mode of transmission of the SARS-CoV-2 USA-WA1/2020 isolate and immunity to SARS-CoV-2 is maintained for at least 56 days. Our studies also indicate protection of the upper respiratory tract against SARS-CoV-2 will require vaccine strategies that mimic natural infection or induce site-specific immunity. Importance The SARS-CoV-2 USA-WA1/2020 strain is a CDC reference strain used by multiple research laboratories. Here, we show the predominant mode of transmission of this isolate in ferrets is by direct contact. We further demonstrate ferrets are protected against re-infection for at least 56 days even when levels of neutralizing antibodies are low or undetectable. Last, we show that when ferrets were vaccinated by the intramuscular route to induce antibodies against SARS-CoV-2, ferrets remain susceptible to infection of the upper respiratory tract. Collectively, these studies suggest protection of the upper respiratory tract will require vaccine approaches that mimic natural infection.

SARS-CoV-2 has initiated a global pandemic and vaccines are being rapidly developed. Using 27 the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the 28 ability of prior infection or vaccine-induced immunity to protect against infection in ferrets.

Ferrets were semi-permissive to infection with the USA-WA1/2020 isolate. When transmission 30 was assessed via the detection of vRNA at multiple timepoints, direct contact transmission was 31 efficient to 3/3 and 3/4 contact animals in two respective studies, while respiratory transmission 32 was poor to only 1/4 contact animals. To assess the durability of immunity, ferrets were re-33 challenged 28 or 56 days post-primary infection. Following viral challenge, no infectious virus 34 was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were 35 several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared.

To determine if intramuscular vaccination protected ferrets against infection, ferrets were In the ferret model of respiratory virus transmission, direct contact transmission is 103 assessed by housing an infected donor animal with a naïve cage-mate, while respiratory 104 transmission, involves housing an infected donor adjacent to a naïve animal (designated 105 respiratory contact) in cages that permit airflow between the animals but prevent direct contact.

Importantly, in this system, large (>5 um) and small droplet (<5 um) transmission cannot be 107 distinguished, and the term respiratory transmission encompasses transmission by airborne 108 particles of both sizes. Since the emergence of SARS-CoV-2, several research groups 109 characterized transmission of different viral isolates in ferrets (14-16, 28, 29) . These groups 110 have reported transmission was efficient (i.e., to 100%) to direct contacts, with variable 111 transmission efficiency to respiratory contacts (i.e., to 33-75% of contacts) (15, 16, 28) .

Therefore, as both contact and respiratory droplet transmission have not been evaluated for the 113 reference strain USA-WA1/2020, we sought to separately evaluate these modes of transmission 114 in ferrets. Moreover, given that ferrets are a highly suitable model of immune-mediated and 

To determine the predominant mode of transmission of the SARS-CoV-2 USA-127 WA1/2020 isolate in ferrets, direct contact and respiratory droplet transmission were evaluated 128 separately. Animals (n=8) were inoculated with 10 5 TCID50 of SARS-CoV-2 in a 1 mL volume. 

For all experimental animals, no overt clinical signs, body weight loss, or elevated 142 temperatures were observed (data not shown). Analyses of levels of vRNA and infectious virus When viral shedding in direct contact ferrets was evaluated ( Figure 1B) , vRNA was detected in 151 all of the contact ferrets at two or more time points. Infectious virus was recovered from one 152 animal on day 3, and a second animal on days 7-11. One contact ferret was excluded from the 153 study due to fighting with its cage mate. Thus, of the 3 direct contact ferrets, all 3 animals had 154 vRNA in the nasal wash, and infectious virus was recovered from 2 of these animals. Analysis 

Collectively, we demonstrate ferrets are semi-permissive to infection with the SARS-

CoV-2 USA-WA1/2020 isolate and that transmission of this isolate is efficient via direct contact 352 and poor by respiratory droplet. We further demonstrate ferrets are protected against re-353 infection for at least 56 days. Using a vaccination strategy to induce serum neutralizing 354 antibodies, ferrets were not protected against SARS-CoV-2 challenge. Thus, while initial human 355 vaccines will likely reduce disease burden, to completely prevent SARS-CoV-2 infections, 356 vaccine strategies that induce immunity in the upper respiratory tract comparable to natural 357 infection will need to be developed.

The CDC reference strain SARS-CoV-2 USA-WA1/2020 was used for all studies. The 

The titer of propagated virus stock was determined on Vero E6 cells in 24-well plates. Nasal 

On day 21 post-viral inoculation and pair housing, the animals were deeply sedated, and 419 blood was collected via cardiac puncture prior to euthanasia with an overdose of sodium 420 pentobarbital. Eight of the donor animals were maintained for re-challenge studies. Blood was 421 collected via the anterior vena cava from these animals, and the animals were given 422 atipamezole and monitored until recovery. Blood samples were processed to recover serum and 423 serum was stored at -80ºC for evaluation of antibody titers.

To evaluate protection against re-infection, 8 donor ferrets from the transmission studies 427 were maintained and blood samples were collected bi-weekly. Blood samples were collected 428 from the anterior vena cava under sedation with ketamine, xylazine and atropine. On day 28 429 and day 56, four ferrets were re-challenged with 10 5 TCID50 as described above. Following 

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Figure 1. Direct contact and respiratory transmission of the SARS-CoV-2 USA

and G-I display three separate transmission studies

each represent a contact transmission study, while panels G-I display data 745 from a respiratory transmission study. Panels A, D, G display nasal wash titers determined by 746 qRT-PCR (left Y-axis) and TCID50 (right Y-axis) for the SARS-CoV-2 inoculated donor animals 747 in each experiment. Line graphs indicate levels of vRNA and bar graphs indicated infectious titers

similarly display nasal wash titers for contact animals. In a given panel, each 749 shaded bar or symbol represents the same animal sampled over multiple time points. Paired 750 donor and contact animals have the same shaded bar or symbol between panels

For all experiments, 4 752 pairs of ferrets (2 pairs of males and 2 pairs of females) were used, and nasal wash samples 753 were collected every other day. Blood was collected on day 21 post-contact. In the first direct 754 contact transmission study, panels A-C, one direct contact animal was removed due to fighting 755 with its cage mate

Viral and antibody titers in ferrets re-challenged with SARS-CoV-2 on day 28 and 56 758 post-primary infection. Panel A and B display nasal wash titers in ferrets re

Line graphs indicate levels of 760 vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right 761 Y-axis) determined via TCID50 on Vero cells. Panel C displays neutralizing antibody titers prior to 762 primary infection