Nirmatrelvir treatment of SARS‐CoV‐2‐infected mice blunts antiviral adaptive immune responses
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Abstract
Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS‐CoV‐2 pandemic. Nirmatrelvir—an orally available inhibitor of the 3‐chymotrypsin‐like cysteine protease—has been shown to reduce the risk of progression to severe COVID‐19. However, the impact of nirmatrelvir treatment on the development of SARS‐CoV‐2‐specific adaptive immune responses is unknown. Here, by using mouse models of SARS‐CoV‐2 infection, we show that nirmatrelvir administration blunts the development of SARS‐CoV‐2‐specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir‐treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.
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Reply to the reviewers
Response to Reviewers' Comments on Fumagalli et al. "Nirmatrelvir treatment blunts the development of antiviral adaptive immune responses in SARS-CoV-2 infected mice" (Preprint RC-2022-01777).
We wish to thank the reviewers for the scholarly review of our work and the very helpful comments. Based on their constructive suggestions, we have generated substantial new experimental data that, in our opinion, positively address all the major and minor concerns raised. In particular, we have confirmed the negative impact of nirmatrelvir treatment on adaptive immune responses in setting of robust SARS-CoV-2 replication (Delta infection in K18-hACE2 transgenic mice …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Response to Reviewers' Comments on Fumagalli et al. "Nirmatrelvir treatment blunts the development of antiviral adaptive immune responses in SARS-CoV-2 infected mice" (Preprint RC-2022-01777).
We wish to thank the reviewers for the scholarly review of our work and the very helpful comments. Based on their constructive suggestions, we have generated substantial new experimental data that, in our opinion, positively address all the major and minor concerns raised. In particular, we have confirmed the negative impact of nirmatrelvir treatment on adaptive immune responses in setting of robust SARS-CoV-2 replication (Delta infection in K18-hACE2 transgenic mice and mouse-adapted SARS-CoV-2 infection of wild-type mice).
One main and one supplemental figure have been added in response to the reviewers' comments. One additional figure – termed Reviewer Figure 1 – has been included in this letter for the reviewers' benefit; while addressing specific comments, we believe that the data depicted in this latter figure remain tangential to the main message of our work and, as such, it should not be incorporated in the final version. To aid the reviewers in the re-evaluation of this study, all relevant passages in the revised text have been written in red. A summary of the changes made to the figures and tables is provided as an appendix at the end of this letter.
Reviewers' comments:
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In this study, Fumagalli et. al evaluated the impact of Nirmatrelvir drug treatment on the development of SARS-CoV-2-specific adaptive immune responses in a mice model. Nirmatrelvir is one of the component of Paxlovid drug that has been shown to reduce the risk of progression to severe COVID-19 and long COVID. Herein, authors show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Upon secondary challenge, nirmatrelvir-treated mice developed fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Overall, the experimental methods, figures, results, statistical analysis and findings of this study are interesting and convincing.
We wish to thank the reviewer for the overall positive assessment of our work.
CROSS-CONSULATION COMMENTS I agree with the Reviewer 2 comments.
__Reviewer #1 (Significance (Required)): __ It was known that nirmatrelvir reduces the risk of severe covid and long covid but, whether its treatment has any impact on adaptive immune response was not known/evaluated. This study has importantly addressed that impact of nirmatrelvir treatment can impair both T and B cell adaptive immune responses. It would have been impactful to understand the mechanism of T and B cell immune response impairment following nirmatrelvir treatment in mice which they have already mentioned a limitation of the study.
We agree with this reviewer that the mechanism of T and B cell impairment following nirmatrelvir treatment should be addressed in future studies.
Moreover this study provides important implications for clinical management of COVID patients and to revise the treatment strategies to avoid virological and/or symptomatic relapse after Paxlovid/nirmatrelvir treatment completion that have been reported in some individuals.
We thank the reviewer for highlighting the impact of our results.
I am not a mice model expert. Not sure whether the viral dose given to mice in this study was optimal to study the impact of the said drug.
Depending on the virus used, we infected mice with 105-106 TCID50. This is in line with most studies of SARS-CoV-2 infection in mice. It is difficult to know what the average infectious dose in humans is, but the human challenge trial in young adults shows that exposure of individuals to as low as 10 TCID50 of SARS-CoV-2 led to detectable viral RNA in the upper airways (Killingley et al, 2022).
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary:
In this manuscript, the authors show that Paxlovid, a commonly used antiviral for SARS-CoV-2 infections, blunts the adaptive immune response to the virus. Indeed, they show convincing effects on T cell and B cell responses in the K18-hACE2 mouse model infected with Omicron variant. The effect is observed when drug treatment was started at 4, 24, or 48 h post infection. Experiments are well done and the data are presented clearly.
We thank the reviewer for the overall positive assessment of our work.
However, the early timing of drug administration resulted in minimal virus replication, thus likely limiting innate immune activation and antigenic exposure. Indeed, the authors show that the drug did not decrease adaptive responses to other viral infections, indicating that the effect on adaptive immunity in SARS-CoV-2 infection can be explained by decreased viral antigen production. Whether this is the mechanism by which relapse infections occur in humans after Paxlovid treatment is unclear.
Major comments:
The authors should discuss whether the timing of drug administration in their experiments is relevant to the timing of when Paxlovid is commonly started in humans. Does Paxlovid limit the adaptive immune response when given later in infection?
We thank the reviewer for raising this valid comment. First, we would like to point out that the kinetics of viral replication upon SARS-CoV-2 exposure differ between mice and humans. When mice are exposed to a high-dose (105 TCID50) aerosolized SARS-CoV-2, they show peak viral replication in the airways at day 3 post exposure and viral RNA is undetectable in the upper and lower airways after day 7 (Reviewer Figure 1A and (Fumagalli et al, 2021)). It is difficult to extract precise data in humans, but the human challenge trial in young adults shows that exposure of individuals to an extremely low dose (10 TCID50!) of SARS-CoV-2 led to the detection of viral RNA in the upper airways for longer than 14 days (Reviewer Figure 1B and (Killingley et al, 2022)). Therefore, it is very difficult to estimate what would possibly mimic what is occurring in treated COVID-19 patients, especially in line of the current COVID-19 guidelines for ritonavir-boosted nirmatrelvir that suggests to initiate treatment as soon as possible and within 5 days of symptoms (https://www.covid19treatmentguidelines.nih.gov/therapies/antiviral-therapy/ritonavir-boosted-nirmatrelvir--paxlovid-/). The choice to start treatment 4 hours after infection was motivated by the original paper that reported in vivo antiviral activity of nirmatrelvir against SARS-CoV-2 (Owen et al, 2021). That said, we performed additional experiments whereby we treated mice with nirmatrelvir 24 or 48 hours after infection (at or near the peak of viral replication). As shown in the new Figure 3, such treatment also resulted in blunted adaptive immune responses.
* *
Reviewer Figure 1. (A) K18-hACE2 mice were exposed to a target dose of 2 x 105 TCID50 of aerosolized SARS-CoV-2 (D614G). Quantification of SARS-CoV-2 RNA in the lung after infection. RNA values are expressed as copy numbers per ng of total RNA and the limit of detection is indicated as a dotted line. (B) Healthy adult volunteers were challenged intranasally with SARS-CoV-2. In the infected individuals (n = 18 biologically independent participants). Viral load in twice-daily nose and throat swab samples was measured by qPCR (blue) and focus-forming assay (red) (a). Results are expressed as mean ± SEM. Adapted from ref. (Killingley et al, 2022).
Omicron variant has limited replication in the K18 mouse model and does not cause disease. Thus, the authors are starting from a model with artificially limited viral antigen production. Does Paxlovid limit the adaptive immune response when given during an infection with a variant strain that replicates robustly in the K18 mice?
We thank the reviewer for raising this issue. In the revised manuscript we have now performed experiments where we infected K18-hACE2 transgenic mice with the Delta (B.1.617.2) variant, known to replicate at higher level compared to the Omicron variants (Shuai et al, 2022). Additionally, we have infected WT mice with a mouse-adapted SARS-CoV-2 (rSARS2-N501YMA30)(Wong et al, 2022) that replicates robustly and induces significant disease. These new results, now shown in the new Figure 3 and new Figure S4, confirm that nirmatrelvir treatment blunts the development of antiviral adaptive immune responses regardless of the variants/strain used for infection.
Reviewer #2 (Significance (Required)):
Significance: Nirmatrelvir/Paxlovid is used clinically for treatment of COVID-19. Relapse infections have been reported after courses of the drug. The authors show here that Paxlovid treatment during a mouse model of SARS-CoV-2 infection results in diminished induction of adaptive immunity and immune memory. This is most probably due to decreased production of viral antigenic stimuli due to inhibition of virus replication. The concept that less viral antigen will result in less induction of immunity is not surprising. Further, whether the phenomenon observed here in a mouse model with poor susceptibility to the chosen virus strain is related to relapse infections in humans was not established. Nonetheless, the audience for this work is broad and this work could be of interest due to the common use of Paxlovid and the ongoing SARS-CoV-2 infections across the world.
Although we did not investigate the mechanism underlying the reported observation in depth, we agree with this reviewer that the most likely explanation for the reduced adaptive immune responses is decreased production of viral antigens. In this regard, it is probably not terribly surprising. However, it is worth noting that successful antimicrobial treatment does not inevitably result in reduced adaptive immune responses to any pathogen. For instance, treatment of mice infected with Listeria monocytogenes with amoxicillin early after infection did not significantly impair the development of T cell responses (Corbin & Harty, 2004; Mercado et al, 2000). Furthermore, treatment with antibiotics before L. monocytogenes infection allowed the development of functional antigen-specific memory CD8+ T cells in the absence of contraction (Badovinac et al, 2004). An additional, and possibly more relevant, example was published during the revision process: monoclonal antibody therapy with bamlanivimab during acute COVID-19 did not impact the development of a robust antiviral T cell response (Ramirez et al, 2022).
As per the comment related the poor susceptibility of the mouse model to the Omicron variants of SARS-CoV-2, we believe that the new data obtained with the Delta variant and with the mouse-adapted SARS-CoV-2 (new Figure 3 and S4) convincingly show that nirmatrelvir treatment blunts antiviral adaptive immune responses to SARS-CoV-2 in mice.
List of modifications
New figures:
- Figure 3: new data as per reviewer’s suggestion.
- Figure S4: new data as per reviewer’s suggestion. __ __
References
Badovinac VP, Porter BB & Harty JT (2004) CD8+ T cell contraction is controlled by early inflammation. Nat Immunol 5: 809–817
Corbin GA & Harty JT (2004) Duration of Infection and Antigen Display Have Minimal Influence on the Kinetics of the CD4+ T Cell Response to Listeria monocytogenes Infection. J Immunol 173: 5679–5687
Fumagalli V, Ravà M, Marotta D, Lucia PD, Laura C, Sala E, Grillo M, Bono E, Giustini L, Perucchini C, et al (2021) Administration of aerosolized SARS-CoV-2 to K18-hACE2 mice uncouples respiratory infection from fatal neuroinvasion. Sci Immunol 7: eabl9929
Killingley B, Mann AJ, Kalinova M, Boyers A, Goonawardane N, Zhou J, Lindsell K, Hare SS, Brown J, Frise R, et al (2022) Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge in young adults. Nat Med 28: 1031–1041
Mercado R, Vijh S, Allen SE, Kerksiek K, Pilip IM & Pamer EG (2000) Early Programming of T Cell Populations Responding to Bacterial Infection. J Immunol 165: 6833–6839
Owen DR, Allerton CMN, Anderson AS, Aschenbrenner L, Avery M, Berritt S, Boras B, Cardin RD, Carlo A, Coffman KJ, et al (2021) An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science 374: 1586–1593
Ramirez SI, Grifoni A, Weiskopf D, Parikh UM, Heaps A, Faraji F, Sieg SF, Ritz J, Moser C, Eron JJ, et al (2022) Bamlanivimab therapy for acute COVID-19 does not blunt SARS-CoV-2-specific memory T cell responses. Jci Insight 7
Shuai H, Chan JF-W, Hu B, Chai Y, Yuen TT-T, Yin F, Huang X, Yoon C, Hu J-C, Liu H, et al (2022) Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron. Nature 603: 693–699
Wong L-YR, Zheng J, Wilhelmsen K, Li K, Ortiz ME, Schnicker NJ, Thurman A, Pezzulo AA, Szachowicz PJ, Li P, et al (2022) Eicosanoid signaling blockade protects middle-aged mice from severe COVID-19. Nature: 1–9
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors show that Paxlovid, a commonly used antiviral for SARS-CoV-2 infections, blunts the adaptive immune response to the virus. Indeed, they show convincing effects on T cell and B cell responses in the K18-hACE2 mouse model infected with Omicron variant. The effect is observed when drug treatment was started at 4, 24, or 48 h post infection. Experiments are well done and the data are presented clearly. However, the early timing of drug administration resulted in minimal virus replication, thus likely limiting innate immune activation and antigenic exposure. Indeed, the authors show that the drug …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors show that Paxlovid, a commonly used antiviral for SARS-CoV-2 infections, blunts the adaptive immune response to the virus. Indeed, they show convincing effects on T cell and B cell responses in the K18-hACE2 mouse model infected with Omicron variant. The effect is observed when drug treatment was started at 4, 24, or 48 h post infection. Experiments are well done and the data are presented clearly. However, the early timing of drug administration resulted in minimal virus replication, thus likely limiting innate immune activation and antigenic exposure. Indeed, the authors show that the drug did not decrease adaptive responses to other viral infections, indicating that the effect on adaptive immunity in SARS-CoV-2 infection can be explained by decreased viral antigen production. Whether this is the mechanism by which relapse infections occur in humans after Paxlovid treatment is unclear.
Major comments:
The authors should discuss whether the timing of drug administration in their experiments is relevant to the timing of when Paxlovid is commonly started in humans.
Does Paxlovid limit the adaptive immune response when given later in infection?
Omicron variant has limited replication in the K18 mouse model and does not cause disease. Thus, the authors are starting from a model with artificially limited viral antigen production. Does Paxlovid limit the adaptive immune response when given during an infection with a variant strain that replicates robustly in the K18 mice?
Significance
Nirmatrelvir/Paxlovid is used clinically for treatment of COVID-19. Relapse infections have been reported after courses of the drug. The authors show here that Paxlovid treatment during a mouse model of SARS-CoV-2 infection results in diminished induction of adaptive immunity and immune memory. This is most probably due to decreased production of viral antigenic stimuli due to inhibition of virus replication. The concept that less viral antigen will result in less induction of immunity is not surprising. Further, whether the phenomenon observed here in a mouse model with poor susceptibility to the chosen virus strain is related to relapse infections in humans was not established. Nonetheless, the audience for this work is broad and this work could be of interest due to the common use of Paxlovid and the ongoing SARS-CoV-2 infections across the world.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
In this study, Fumagalli et. al evaluated the impact of Nirmatrelvir drug treatment on the development of SARS-CoV-2-specific adaptive immune responses in a mice model. Nirmatrelvir is one of the component of Paxlovid drug that has been shown to reduce the risk of progression to severe COVID-19 and long COVID. Herein, authors show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Upon secondary challenge, nirmatrelvir-treated mice developed fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Overall, the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
In this study, Fumagalli et. al evaluated the impact of Nirmatrelvir drug treatment on the development of SARS-CoV-2-specific adaptive immune responses in a mice model. Nirmatrelvir is one of the component of Paxlovid drug that has been shown to reduce the risk of progression to severe COVID-19 and long COVID. Herein, authors show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Upon secondary challenge, nirmatrelvir-treated mice developed fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Overall, the experimental methods, figures, results, statistical analysis and findings of this study are interesting and convincing.
Referees cross-commenting
I agree with the Reviewer 2 comments.
Significance
It was known that nirmatrelvir reduces the risk of severe covid and long covid but, whether its treatment has any impact on adaptive immune response was not known/evaluated. This study has importantly addressed that impact of nirmatrelvir treatment can impair both T and B cell adaptive immune responses. It would have been impactful to understand the mechanism of T and B cell immune response impairment following nirmatrelvir treatment in mice which they have already mentioned a limitation of the study.
Moreover this study provides important implications for clinical management of COVID patients and to revise the treatment strategies to avoid virological and/or symptomatic relapse after Paxlovid/nirmatrelvir treatment completion that have been reported in some individuals.
I am not a mice model expert. Not sure whether the viral dose given to mice in this study was optimal to study the impact of the said drug.
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