SARS-CoV-2 infection induces protective immunity and limits transmission in Syrian hamsters

Using an aged Syrian hamster model, which develops severe morbidity upon SARS-CoV-2 infection including death, we demonstrated that the immunity induced by a natural infection not only protects hamsters from reinfection but also significantly limits transmission via close contact.

Lien et al is a well written study describing a model of SARS-CoV-2 infection in young and aged Syrian hamsters. This is similar to recently published Imai et al, which showed Syrian hamsters as a potential model for Sars-CoV-2 infection. Uniquely, the authors show that rechallenged hamsters could not transmit the virus, and is severely impaired from transmitting the virus. This is especially important for public health policies and attention should be brought to it.
Major comments: 1) I am unclear how neutralizing antibody titers were measured ( Figure 1O). Was this by PRNT? In this figure, was it only for the animals infected with 100TCID50? How about PRNT for animals infected with other titers? Neutralization assay is missing from the methods.
2) "Contact hamster developed viral pneumonia and seroconverted despite no observed weight loss" -Was there any viral load in the lung as measured by plaque assay or TCID50?
3) Recent studies by Song et al showed neuroinvasion of Sars-CoV-2 in human brain. Similarly, Imai et al found virus in the brains of infected hamsters. Did the authors find virus in the brains of the young hamsters ( Figure 1E)? More importantly, did the authors check for brain viral loads in older hamsters that succumbed to the lethal infection? This could potentially lead to a model to understand why some patients present with viral neuroinvasion. 4) Clinically, patients with severe disease have been shown to present with a cytokine storm. Was there any indication of a cytokine storm (either by luminex or transcriptomics) in the infected animals? 5) Viral RNA that was detected in the lungs ( Figure 3E) is unlikely to be from the input challenge virus 7 days post infection. Viruses would have been cleared or degraded at this point. 4 logs of virus is quite high, can the authors comment further on this? 6) All viral loads in the manuscript were measured by RT-PCR. To show that these are infectious virions, can the authors provide plaque titers or TCID50 titers? 7) Recent studies have indicated an important role of T cells in providing protection against infection, did the authors measure T cell responses in the hamsters in the re-infection model? Perhaps the authors can comment on this in the discussion.
Minor Comments: 1) The manuscript lacked page or lined numbers which makes comments challenging. Could the authors add this in during the revision?
2) It would be helpful if the authors could use arrows to indicate on their H&E histology slides immune infiltrates, necrosis, loss of cilia or edema that is observed. Zoomed in images of the pathology would further aid any readers to interpret the images.
3) Dot plots in the figures have points below the threshold. Visually, it might be easier if the authors floor the negative values to baseline. 4) With respect to the abstract and discussion, another lethal infection of an immunocompetent mouse model was uploaded to bioarchive (de alwis et al). The authors can instead claim that they are the first lethal hamster model?
In this study, Lien and colleagues developed a Syrian hamster animal model for COVID-19 research studies. In agreement with previously reported studies, the authors show that aged hamsters experienced more pronounced signs of disease and more consistent weight loss than young hamsters. Infected hamsters were protected from a second challenge 28 days after the primary infection, and did not infect naïve animals. This work is mostly a confirmatory study as other groups have previously reported similar findings in greater detail. Some of the data provided are very preliminary and are not strong enough to draw significant conclusion.
Specific Issues: 1.Viral titers should be determined by plaque assay or TCID50 and not only by qRT-PCR.
2.IHC analysis for detection of viral antigens would be a nice addition to the histopathology analysis.
3.In figure 2 the authors compare one or two animals/group. Increasing the number of animals in the study, and including a more detailed analysis of virus replication in different organs would help strengthening their conclusions. A full necropsy evaluation of the deceased hamster should have been performed to understand whether this death was related to SARS-CoV-2 infection or not. Figure 3 and 4 lack important controls. Figure 3 doesn't include a mock infection control for the primary infection. Figure 4 doesn't include a positive control for transmission.

Appeal Request
October 26, 2020 Dear Dr. Bhatt, The authors of manuscript #LSA-2020-00886-T have requested an appeal. Their comments are below.
Dear Editor, We are submitting a revised manuscript titled "SARS-CoV-2 infection induces protective immunity in the lung and limits transmission in Syrian hamsters" (LSA-2020-00886-T) for consideration of publication in Life Science Alliance.
The initial review, while recognizing the topic of the study is of interest to Life Science Alliance, raised a series of concerns. As summarized by the editor, "Although your manuscript is intriguing, I feel that the points raised by the reviewers are more substantial than can be addressed in a typical revision period". We are pleased to report that we have accumulated more data while waiting for the review comments. In the revised manuscript, we have included the following results to strengthen our conclusions: 1. HE slides of brain and olfactory bulb from uninfected and infected hamsters (revised Fig. S1) 2. Detection of viral genomic RNA in the lung of infected hamsters either through contact transmission or direct inoculation (revised Fig. S2I.J and Fig. 2G-I).
3. Additional lethality in aged hamsters (revised Fig. 2J). 4. Virus titers in nasal washes (measured by TCID50 assay) were added to Fig. 3E. 5. Positive control for transmission-mediated infection (revised Fig. 4C) A point-by-point response to the referee comments is also attached to this submission.
We look forward to a favorable review on this very important study. Thank you! Tony You can accept or decline this request from the manuscript using the following link: We have considered your appeal for our decision on "SARS-CoV-2 infection induces protective immunity in the lung and limits transmission in Syrian hamsters". After assessing the revised manuscript, point-by-point rebuttal and your appeal letter, the academic editors and I are pleased to let you know that we have decided to send your manuscript for external review.
Please use the following link to submit your revised manuscript: https://lsa.msubmit.net/cgibin/main.plex?el=A4Na6Wy3A3Cjtu4I1B9ftdYS3aDNv9d34WwZ6boTBsPgZ We will let you know when the reviews have been received and a decision has been made.
Yours sincerely, Shachi Bhatt, Ph.D. Executive Editor Life Science Alliance https://www.lsajournal.org/ Tweet @SciBhatt @LSAjournal 1st Author's response to Reviewers December 14, 2020 The revision contains the following additional new results to strengthen our conclusions: 1. HE slides of brain and olfactory bulb from uninfected and infected hamsters (revised A point-by-point response to the referee comments is provided as below (original review comments in blue and our response in black): Reviewer: 1 1-I am unclear how neutralizing antibody titers were measured ( Figure 1O). Was this by PRNT? In this figure, was it only for the animals infected with 100TCID50? How about PRNT for animals infected with other titers? Neutralization assay is missing from the methods.
Our response: The neutralizing antibody titers (nABs) shown in Figure 1O were determined using a SARS-CoV-2 pseudovirus neutralization assay, which had been described in the Materials and Methods section. This is an assay that we have qualified here at FDA with good correlation with titers obtained from the standard PRNT. The nAB titers shown in Figure 1O included nine hamsters that were challenged with 10 5 TCID 50 (shown in black solid circles) as well as those with 100 and 10 TCID 50 in blue and green circles, respectively.
2-"Contact hamster developed viral pneumonia and seroconverted despite no observed weight loss" -Was there any viral load in the lung as measured by plaque assay or TCID50?
Our response: We did not save the lung tissue of this particular "contact hamster" for plaque assay. However, we did perform RNAscope to show the presence of viral genomic RNA in the infected lung ( Fig. S2I-J). . It must be pointed out, however, SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality in this transgenic mouse model. By contrast, patients who succumb to SARS-CoV-2 infection typically die of acute respiratory distress syndrome (ARDS). To the best of our knowledge, most groups who work with the hamster model have not been able to cultivate live infectious virus from the brains of hamsters that are challenged with SARS-CoV-2. We have repetitively examined brains, including the olfactory bulbs of infected hamsters and could not find any significant lesion. Having said that, we cannot rule out the possibility that a transient low degree infection may occur in the olfactory bulb or even in the brain. Nonetheless, HE images and RNAscope images from uninfected and infected hamster brains can be found in the Our response: This is an excellent question that is worth further investigation. One of the caveats working with Syrian hamsters is the relative lack of reagents for conducting immunological assays. To the best of our knowledge, there is no luminex assay available for hamsters to directly measure cytokines. Transcriptomics analyses of infected hamster tissues do show the upregulation of cytokine genes (Benjamin R. tenOever et al), although it is unclear whether infection induces a cytokine storm.

3-Recent studies by
With the aged hamster model in hand, we are exploring several techniques to investigate along this line and hopefully will able to provide an update in the future.
5-Viral RNA that was detected in the lungs ( Figure 3E) is unlikely to be from the input challenge virus 7 days post infection. Viruses would have been cleared or degraded at this point. 4 logs of virus is quite high, can the authors comment further on this?
Our response: we thank the reviewer for the comment. We typically see 7-10 logs of virus in the infected lungs, so 3-4 logs of virus (vRNA titers) was indeed quite low given that the genomic RNA/PFU ratio of SARS-CoV-2 is about 1000-10,000 as reported by Plante et al. In other words, 3-4 logs of virus (in vRNA titers) may have only 1-10 infectious virus particles. Nonetheless, we deleted the phrase "likely be from the input challenge virus" in the revision.
6-All viral loads in the manuscript were measured by RT-PCR. To show that these are infectious virions, can the authors provide plaque titers or TCID50 titers?
Our response: We have updated Figure 3E to include TCID 50 titers in nasal washes.
7-Recent studies have indicated an important role of T cells in providing protection against infection, did the authors measure T cell responses in the hamsters in the re-infection model? Perhaps the authors can comment on this in the discussion.
Our response: we have not measured T cell responses in hamsters, primarily because of lacking reagents. Nonetheless, we have attempted to block T cell response using a commercial antibody and have seen extended period of infection in hamsters. Jay Hooper's group at USAMRIID reported that Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters developed clinical signs of disease that were more severe than in immunocompetent hamsters upon SARS-CoV-2 challenge, suggesting that functional B and/or T cells are likely to play an important role for the clearance of SARS-CoV-2 and in protection from acute disease.

Minor comments
1) The manuscript lacked page or lined numbers which makes comments challenging. Could the authors add this in during the revision? Our response: We thank the reviewer for the advice and have included page and line numbers to the revised manuscript.
2) It would be helpful if the authors could use arrows to indicate on their H&E histology slides immune infiltrates, necrosis, loss of cilia or edema that is observed. Zoomed in images of the pathology would further aid any readers to interpret the images.
Our response: We thank the reviewer for the advice and have added indicators for histopathology changes and a closeup image (Fig. S1L). We would also like to kindly remind that there are several hundred HE slides related to this study and many of them cannot be included in the manuscript due to space constraint. Nonetheless, all HE images that are presented in the manuscript are captured in high resolution, meaning that the readers should be able to Zoom in easily and see details as they wish.
3) Dot plots in the figures have points below the threshold. Visually, it might be easier if the authors floor the negative values to baseline. Our response: We amended all figures as the reviewer recommended. 4) With respect to the abstract and discussion, another lethal infection of an immunocompetent mouse model was uploaded to bioarchive (de alwis et al). The authors can instead claim that they are the first lethal hamster model? Our response: De alwis et al were using the hACE2 transgenic mouse model, which develops lethality after SARS-CoV-2 infection due to neuroinvasion, but not respiratory infection. This finding has been documented by multiple groups. To distinguish our model from the transgenic mouse model, we have now amended the relevant text to read "the first lethal model using genetically unmodified laboratory animals".
Reviewer: 2 Comments to the Author In this study, Lien and colleagues developed a Syrian hamster animal model for COVID-19 research studies. In agreement with previously reported studies, the authors show that aged hamsters experienced more pronounced signs of disease and more consistent weight loss than young hamsters. Infected hamsters were protected from a second challenge 28 days after the primary infection and did not infect naïve animals. This work is mostly a confirmatory study as other groups have previously reported similar findings in greater detail. Some of the data provided are very preliminary and are not strong enough to draw significant conclusion.
Our response: We were among the earliest groups who started the Syrian hamster model for SARS-CoV-2. Most results included in the manuscript were first presented to a World Health Organization working group on July 9, 2020. The intention of our study is not to confirm what other have reported. The novelty of this study resides in twofold: 1) we have established a lethal animal model for COVID-19. This is very significant because the only other animal model that sometimes shows lethality is the K18 hACE2 transgenic mouse model. The cause of death in a fraction of SARS-CoV-2 challenged hACE2 transgenic mice is neuroinvasion, which differs from severe COVID-19 cases where patients mainly die of ARDS. Using aged hamsters without any genetic modification, we consistently observed lethality. This aged hamster model not only allows investigation of the pathogenesis of severe and even fatal COVID-19 cases, but also represents a superior platform for evaluating efficacies of vaccines and antivirals.
2) This study attempted to address one of most important questions regarding SARS-CoV-2, i.e., prior infections will likely protect from a secondary SARS-CoV-2 infection, but will it prevent transmission? Illustrated in the figure below, when a convalescent individual (Subject A) becomes re-exposed to a virus carrier (the Spreader), there are likely two scenarios: 1, the immunity that Subject A has acquired from prior infection not only protects him/her from getting pneumonia, but also prevents transmission to other naïve individuals because Subject A sheds no or very little virus. Under such a scenario, the spread of the virus through Subject A is virtually stopped. 2, Subject A is protected from developing severe diseases but continues to shed virus because the acquired immunity is not strong enough to curtail the replication of virus in the nose or upper respiratory tract. In this case, Subject A serves as a virus spreader.
Our results clearly demonstrated that scenario #1 can be achieved in the hamster model. Ongoing research in the laboratory is to examine whether a vaccine would accomplish the same. Such information is critical for decision-making of public health policies and evaluating efficacy of vaccine candidates. There have been no published studies that directly address this important question.
Lastly, we provided additional data to strengthen the conclusions. We apologize that perhaps because we have worked with the hamsters for so long and hence have taken many findings for granted. For example, we have done the transmission study many times and it works consistently, however, this information is not obvious to the reviewer. Therefore, we may have skipped some controls that the reviewer deems essential.
Specific Issues: 1-Viral titers should be determined by plaque assay or TCID50 and not only by qRT-PCR. Our response: We have updated Figure 3E to include TCID50 titers in nasal washes.
2 -IHC analysis for detection of viral antigens would be a nice addition to the histopathology analysis. Our response: In the revised manuscript, we have included RNAscope data to demonstrate the infection of lungs.
3 -In figure 2 the authors compare one or two animals/group. Increasing the number of animals in the study and including a more detailed analysis of virus replication in different organs would help strengthening their conclusions. A full necropsy evaluation of the deceased hamster should have been performed to understand whether this death was related to SARS-CoV-2 infection or not.
Our response: To clarify, in the experiment shown in Figure 2, we had 20 hamsters for the 10 5 TCID 50 , 2 for 10 4 , 1000, 100 and 10 TCID 50 group. Not all hamsters were sacrificed on the same day or at the end of the study, therefore we only plotted graphs using matched samples. Therefore, the readers see fewer data points in Figure 2. Furthermore, we have challenged hamsters using different doses in several Subject A Spreader different experiments and obtained similar observations, but because these experiments were conducted on different dates, we did not pool results from those hamsters in the graphs. Nonetheless, the conclusions remain the same.
Regarding the cause of the death of the deceased hamster (Fig. 2), a full necropsy indeed was performed. Lesions are not observed in the pancreas, small intestine, large intestine, brain. The animal had severe, diffuse bronchointerstitial pneumonia along with myocardial disease and thrombus formation in the left atrium, leading to death of this animal. RNAscope images are now provided to show the presence of viral genomic RNA in the infected hamster lung but not in the brain (Fig. 2G-I). Figure 3 and 4 lack important controls. Figure 3 doesn't include a mock infection control for the primary infection. Figure 4 doesn't include a positive control for transmission.

-The experiments described in
Our response: We started working with the hamster model as early as March 2020 and the experimental procedures have been optimized to ensure reproducibility. For example, the same lot of virus has been used up to date for inoculation; inoculated hamsters always develop pneumonia with measurable viral loads in the lung ranging between 10 7 and 10 9 copies/0.1g; transmission-mediated infection of hamsters always occurs under the current protocol. Such information, although seems routine to us, were not mentioned in the initial submission which might have prompted the reviewer to think that the experiments described in Figure 3 and 4 lack important controls.
Particularly, hamsters in Figure 3 were previously infected at the same time with those hamsters shown in Figure 1. The re-infection was done under the same procedure using the same lot of virus. Also, at the time when we were performing the experiment reported in Figure 3, another project that was being simultaneously conducted in the laboratory did include hamsters that could serve as mock infection control. These hamsters readily developed pneumonia upon challenge and showed much higher viral loads in the lung. Because the results are intended for another project, we could not include them as a mock infection control group as the reviewer would like to see. Nonetheless, the results and conclusions of Figure 3 are well supported.
Same thing could be said to Figure 4. The procedure has been established to provide transmission at 100% success rate. To satisfy the reviewer, we now included data points of two contact hamsters that were exposed infected hamsters as positive controls for transmission (revised Fig. 4C).
In summary, we believe the revised manuscript has been improved significantly and hope these changes will warrant a positive decision. Thank you for submitting your revised manuscript entitled "SARS-CoV-2 infection induces protection in the lung and limits transmission in Syrian hamsters". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet the minor text edits requested by Rev 1 and our formatting guidelines.
We apologize for this delay in getting back to you, one of the reviewers took longer than expected to send us their re-review.
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