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.
ResultsBy 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.
ImpactWhile the clinical data continue to support nirmatrelvir treatment for the prevention of severe COVID-19 in high-risk individuals, the data reported here draw attention to a potential negative impact of this therapy. We believe that our results might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals and should inform clinical and public health policies.
The COVID-19 outbreak, caused by SARS-CoV-2, has resulted in more than 670 million confirmed infections causing greater than 6.8 million deaths worldwide as of February 2023 (
Paxlovid, a combination of an orally available Mpro inhibitor termed nirmatrelvir (Owen et al, 2021) and ritonavir, has received an Emergency Use Authorization (EUA) from the Food and Drug Administration (FDA) for the treatment of COVID-19 on December 22, 2021. Despite its effectiveness at reducing viral titers and the risk of progressing to severe COVID-19 (Hammond et al, 2022; Najjar-Debbiny et al, 2022), the impact of nirmatrelvir treatment on the development of adaptive immunity to SARS-CoV-2 is unknown. It is also unclear why some patients experience a surprising rebound of viral load and a rapid relapse of COVID-19 symptoms shortly after the completion of an early and effective nirmatrelvir treatment (Boucau et al, 2022; preprint: Dai et al, 2022; Gupta et al, 2022; Ranganath et al, 2022; Soares et al, 2022; preprint: Charness et al, 2022a; Charness et al, 2022b; preprint: Wang et al, 2022a,b). Viral sequencing indicates that the relapse is not associated with the selection of treatment-resistant mutations, or due to infection with different SARS-CoV-2 variants (Boucau et al, 2022; Gupta et al, 2022). Whether such rebound is part of the natural history of COVID-19 or is due to an impairment in the development of adaptive immunity necessary to complete SARS-CoV-2 clearance remains to be determined.
Results and DiscussionHere, we set out to assess the impact of nirmatrelvir treatment on the development of antiviral adaptive immunity in a well-characterized mouse model of COVID-19 based on the controlled administration of aerosolized SARS-CoV-2 to K18-hACE2 transgenic mice (Fumagalli et al, 2021).
Nirmatrelvir was synthesized prior to its publication (Owen et al, 2021) via a multistep convergent approach that differed from the reported procedure (Fu et al, 2020; Jin et al, 2020; Cannalire et al, 2022). Details and synthesis intermediates are shown in Fig 1A and Materials and Methods. As expected, nirmatrelvir was able to inhibit the activity of the SARS-CoV-2 Mpro in a dose-dependent manner, as determined by a fluorescence resonance energy transfer (FRET)-based biochemical assay performed both by preincubating the protease with the compound for 30 min at 37°C (Fig 1B) or by directly adding the substrate to the reaction (Appendix Fig S1A), as described (Owen et al, 2021). In the two assays, the 50% inhibitory concentration (IC50) of nirmatrelvir on the Mpro was 47 nM (Fig 1B) and 14 nM (Appendix Fig S1A), respectively. In both procedures, we used the commercially available SARS-CoV-2 Mpro inhibitor GC376 (Fu et al, 2020; Ma et al, 2020; Cannalire et al, 2022; Luan et al, 2022) as positive control, showing an IC50 of 0.14 nM (Fig 1B) and 4.8 nM (Appendix Fig S1A), respectively. Next, the antiviral activity of nirmatrelvir against SARS-CoV-2 was examined by monitoring the protection from cytopathic effect in infected HEK293T-hACE2 cells (Fig 1C) and by quantifying SARS-CoV-2 RNA in the supernatant (Appendix Fig S1B). Nirmatrelvir prevented death of HEK293T-hACE2 cells infected with the SARS-CoV-2 variants D614G, B.1.617.2 (Delta), and B.1.1.529 (Omicron BA.1) with a mean IC50 value of 33 ± 10 nM (Fig 1C) and prevented SARS-CoV-2 RNA release with a mean IC50 value of 54 ± 25 nM (Appendix Fig S1B). Finally, we evaluated the antiviral activity of nirmatrelvir in a well-characterized mouse model of COVID-19, based on the controlled administration of aerosolized SARS-CoV-2 to K18-hACE2 transgenic mice (Fumagalli et al, 2021). Briefly, non-anesthetized mice were placed in a nose-only inhalation tower system and exposed to a target dose of 2 × 105 tissue culture infectious dose 50 (TCID50) of aerosolized B.1.1.529 under controlled pressure, temperature, and humidity conditions (Fig 1D). Mice were treated six times with vehicle or nirmatrelvir via oral gavage (150 mpk/mouse) starting at 4 h post-infection (p.i.) and every 12 h thereafter up until day 3 p.i. (Fig 1D). The plasma concentration of nirmatrelvir evaluated 4 h after the last administration was 1.39 ± 0.73 μM (Appendix Fig S1C). As expected, neither SARS-CoV-2 infection nor nirmatrelvir treatment affected the body weight of K18-hACE2 mice (Fig 1E). In line with previously published studies (Fumagalli et al, 2021; Owen et al, 2021), whereas vehicle-treated SARS-CoV-2-infected mice showed robust viral replication in the lungs and in the nasal turbinates, nirmatrelvir-treated SARS-CoV-2-infected mice showed virtually undetectable viral RNA and infectious virus in the same anatomical compartments (Fig 1F and G).
FigureSynthesis of nirmatrelvir and characterization of its biochemical and antiviral activity 1. Synthesis of nirmatrelvir. Reagents and conditions: (i) HBTU, DIPEA, dry CH2Cl2, room temperature (RT), 16 h, 78%; (ii) 1 N aq. LiOH/THF (1:1), RT, 2 h, 100%; (iii) HBTU, DIPEA, dry CH2Cl2/DMF, RT, 3 h, 75%; (iv) 4 N HCl in 1,4-dioxane/CH2Cl2 (1:1), 0°C to RT, 2 h, 100%; and (v) a: TFAA, dry Py, dry CH2Cl2, 0°C to RT, 2 h; b: TFAA, dry Py, dry CH2Cl2, 0°C to RT, 15 h, 40% over a and b.Dose-dependent inhibition of nirmatrelvir (left panel) and GC376 (right panel) on SARS-CoV-2 Mpro. Prior to adding the substrate for the biochemical reaction, the protease was preincubated for 30 min at 37°C with the indicated concentrations of the compound.Dose-dependent antiviral activity of nirmatrelvir in HEK293T-hACE2 cells infected with SARS-CoV-2 D614G (purple symbols), B.1.617.2 (green symbols), or B.1.1.529 (blue symbols). Antiviral activity was determined as percent inhibition of the virus-induced cytopathic effect.Schematic representation of the experimental setup. Non-anesthetized K18-hACE2 transgenic mice were infected with a target dose of 2 × 105 TCID50 of SARS-CoV-2 B.1.1.529 through aerosol exposure (see Materials and Methods for details). Infected mice were treated with 150 mg/kg (mpk) of nirmatrelvir (red symbols, n = 4) or vehicle (blue symbols, n = 4) six times by oral gavage (P.O.) starting 4 h post-infection (p.i.), and every 12 h thereafter. Mock-treated mice were used as control (black symbols, n = 3). Lung, nasal turbinates, and blood were collected and analyzed 3 days p.i.Mouse body weight was monitored daily and is expressed as the percentage of weight relative to the initial weight.Quantification of SARS-CoV-2 RNA (left panel) and viral titers (right panel) in the lung 3 days after infection. RNA values are expressed as copy number per ng of total RNA and the limit of detection is indicated as a dotted line. Viral titers were determined by median tissue culture infectious dose (TCID50).Quantification of SARS-CoV-2 RNA in the nasal turbinates 3 days after infection. RNA values are expressed as copy number per ng of total RNA and the limit of detection is indicated as a dotted line.Data information: Data in B and C represent the mean ± SD of three biological replicates and are representative of three technical replicates; data in E–G are expressed as mean ± SEM and are representative of at least two independent experiments. *P-value [less than] 0.05, **P-value [less than] 0.01; two-way ANOVA followed by Sidak's multiple-comparison test (E); Kruskal–Wallis test followed by uncorrected Dunn's test, each comparison stands alone (F and G).Source data are available online for this figure.
With this established system, we next set out to study the consequences of nirmatrelvir treatment on antiviral immune responses. To this end, we infected and treated another group of K18-hACE2 transgenic mice exactly as before but monitored them until 24 days p.i. to assess the SARS-CoV-2-specific antibody response in the sera. Because SARS-CoV-2 T cells are not readily detectable in the blood of K18-hACE2 transgenic mice infected with B.1.1.529 (Appendix Fig S2A and B), we decided to subject the mice to a homologous re-challenge with a higher dose (1 × 106 TCID50) of aerosolized SARS-CoV-2 B.1.1.529 to evaluate the eventual recruitment of memory T (and B) cells to the infected lung and lung-draining mediastinal lymph nodes (Fig 2A). The mean plasma concentration of nirmatrelvir 4 h after the last administration was 1.40 ± 0.99 μM (Appendix Fig S2C), remarkably similar to the previous experiment. No mice exhibited significant weight loss for the whole duration of the experiment (Fig 2B). Of note, the levels of total IgG specific for the spike S1 subunit (receptor binding domain, RBD) (Fig 2C) and the levels of anti-B.1.1.529-neutralizing antibodies (Fig 2D) were remarkably reduced in nirmatrelvir-treated mice 14 and 21 days p.i., respectively, and 4 days after re-challenge (Fig 2E and F). Consistent with this, B cells recovered from the mediastinal lymph nodes of nirmatrelvir-treated mice 4 days after re-challenge exhibited a lower expression of the activation marker CD95 (Fig 2G), and in one vehicle-treated mouse, we could detect RBD-specific B cells (Fig 2H). Additionally, large lymphocytic aggregates consisting of proliferating B cells were detected in the lungs of vehicle- but not nirmatrelvir-treated mice, 4 days after re-challenge (Appendix Fig S3). SARS-CoV-2-specific CD8+ and CD4+ T cells recovered from lung homogenates were assessed for intracellular IFN-γ and TNF-α expression upon in vitro stimulation with a pool of SARS-CoV-2 peptides covering the complete nucleocapsid, membrane, and spike proteins (Silva-Cayetano et al, 2021). In line with the results obtained for the humoral response, we found that the frequency and absolute number of IFN-γ+ and IFN-γ+ TNF-α+ SARS-CoV-2-specific CD8+ and CD4+ T cells were significantly lower in the lungs of nirmatrelvir-treated mice compared to vehicle-treated mice, 4 days after homologous re-challenge (Fig 2I–N). Immune responses to SARS-CoV-2 were also reduced when nirmatrelvir treatment was initiated 24 and 48 h after exposure to aerosolized B.1.1.529 (Fig 3A): indeed, we observed a significant reduction in the anti-S1 RBD IgG levels 14 and 21 days p.i. (Fig 3B) and 4 days after heterologous re-challenge with SARS-CoV-2 Omicron subvariant BA.5 (Fig 3C). Similarly, K18-hACE2 transgenic mice infected with the more pathogenic Delta (B.1.617.2) variant (Shuai et al, 2022) showed a reduction in humoral responses to SARS-CoV-2 upon nirmatrelvir treatment (Fig 3D and E). Moreover, the expression of the activation marker CD44+ on CD8+ T cells (Fig 3F) and the frequency of granzyme-B+ and IFN-γ+ CD8+ T cells were reduced in nirmatrelvir-treated mice compared to vehicle-treated mice (Fig 3G and H). Finally, the negative impact of nirmatrelvir treatment on immune responses to SARS-CoV-2 were also evaluated in C57BL/6 mice infected with a mouse-adapted SARS-CoV-2 (rSARS2-N501YMA30) (Wong et al, 2022) which replicates more robustly and is more pathogenic than B.1.1.529, yet maintains its sensitivity to nirmatrelvir (Appendix Fig S4A–D). Consistent with previous results, the levels of total IgG specific for the spike RBD were remarkably reduced in C57BL/6 mice that were treated with nirmatrelvir 1 day after infection with rSARS2-N501YMA30 (Fig 3I and J).
FigureImpact of nirmatrelvir treatment on adaptive immune responses to SARS-CoV-2 2. ASchematic representation of the experimental setup. Non-anesthetized K18-hACE2 mice were exposed to a target dose of 2 × 105 TCID50 of aerosolized SARS-CoV-2 B.1.1.529 (see Materials and Methods for details). Infected mice were treated with 150 mpk of nirmatrelvir (red symbols, n = 5) or vehicle (blue symbols, n = 6) for six times by oral gavage (P.O.) starting 4 h p.i., and every 12 h thereafter. Twenty-four days after infection, mice were re-challenged with a target dose of 1 × 106 TCID50 of SARS-CoV-2 B.1.1.529 through aerosol exposure. Mock-treated mice were used as non-infected controls (black symbols, n = 3). A group of naïve mice challenged with 1 × 106 TCID50 of SARS-CoV-2 B.1.1.529 served as additional controls (green symbols, n = 5). Blood was collected 7, 14, and 21 days after the first infection and 4 days post-re-challenge. Lung, nasal turbinates, and lung-draining mediastinal lymph nodes (mLN) were collected and analyzed 4 days post-re-challenge.BMouse body weight was monitored daily and is expressed as percentage of weight relative to the initial weight.C–FQuantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice (C) 7, 14, and 21 days p.i. or (E) 4 days post-re-challenge. Neutralization dose 50 (ND50) against SARS-CoV-2 B.1.1.529 pseudovirus in the plasma of the indicated mice (D) 7, 14, and 21 days p.i. or (F) 4 days post-re-challenge.GFlow cytometry histogram (left panel) and percentage (right panel) of B cells stained positive for CD95 in the mLN of the indicated mice 4 days post-re-challenge (pre-gated on live+/CD4−/ CD8−/ B220+/CD19+ cells).HFlow cytometry plot of RBD-specific B cells detected by two fluorescently labeled streptavidin-based RBD-biotinylated tetramers in the mLN of one vehicle-treated mouse 4 days post-re-challenge (pre-gated on live+/CD4−/ CD8−/ B220+/CD19+ cells).I–NRepresentative flow cytometry plots of (I) CD8+ T cells or (L) CD4+ T cells expressing IFN-γ and TNF-α in the lungs of the indicated mice 4 days post-re-challenge. Unstimulated cells are shown in the upper panels, whereas cells re-stimulated with a pool of SARS-CoV-2 peptides for 4 h at 37°C are shown in the bottom panels. Plots were pre-gated as (I) live+/B220−/CD19−/CD4−/CD8+ cells or (L) live+/B220−/CD19−/CD8−/CD4+. Frequency (J, M) and absolute number (K, N) of IFN-γ- and TNF-α-producing CD8+ T cells (J, K) or CD4+ T cells (M, N) in the lung of the indicated mice 4 days post-re-challenge.Data information: Data are expressed as mean ± SEM and are representative of at least two independent experiments. *P-value [less than] 0.05, **P-value [less than] 0.01, ***P-value [less than] 0.001; two-way ANOVA followed by Sidak's multiple-comparison test (B-D); Kruskal–Wallis test followed by uncorrected Dunn's test, each comparison stands alone (E); Mann–Whitney test (F); and one-way ANOVA followed by uncorrected Fisher's LSD, each comparison stands alone (G, J, K, M, N). Normal distribution was verified by Shapiro–Wilk test.Source data are available online for this figure.
FigureNirmatrelvir treatment of SARS-CoV-2-infected mice blunts the development of antiviral adaptive immune responses independent of timing of treatment and viral variant 3. ASchematic representation of the experimental setup. Non-anesthetized K18-hACE2 mice were exposed to a target dose of 2 × 105 TCID50 of aerosolized SARS-CoV-2 B.1.1.529. Infected mice were treated with 150 mpk of nirmatrelvir (red symbols) or vehicle (blue symbols, n = 4) six times by oral gavage (P.O.) starting 24 h p.i. (full-red symbols, n = 5) or 48 h p.i. (empty-red symbols, n = 5), and every 12 h thereafter. Thirty-eight days after infection, mice were re-challenged with a target dose of 1 × 106 TCID50 of SARS-CoV-2 BA.5 through aerosol exposure. Mock-treated mice were used as non-infected controls (black symbols, n = 3).B, CQuantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice (B) 14 and 21 days p.i. and (C) 4 days post-re-challenge.DSchematic representation of the experimental setup. Non-anesthetized K18-hACE2 mice were exposed to a target dose of 2 × 105 TCID50 of aerosolized SARS-CoV-2 B.1.617.2. Infected mice were treated with 150 mpk of nirmatrelvir (red symbols, n = 3) or vehicle (blue symbols, n = 4) for six times by oral gavage (P.O.) starting 24 h p.i., and every 12 h thereafter. Mock-treated mice were used as non-infected controls (black symbols, n = 2).EQuantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice 7 days p.i.FFlow cytometry histogram (left panel) and geometric mean fluorescent intensity (gMFI) quantification (right panel) of CD44 expression by CD8+ T cells in the blood of the indicated mice 7 days p.i. (pre-gated on live+/ B220−/ CD19−/ CD4−/ CD8+ cells).G, HFrequency of (G) granzyme-B- and (H) IFN-γ-producing CD8+ T cells in the blood of the indicated mice 7 days p.i. Cells were stimulated in vitro with a pool of SARS-CoV-2 peptides for 4 h at 37°C. Plots were pre-gated as above.ISchematic representation of the experimental setup. Non-anesthetized C57BL/6 mice were exposed to a target dose of 1 × 105 TCID50 of aerosolized rSARS-N501YMA30. Infected mice were treated with 150 mpk of nirmatrelvir (red symbols, n = 4) or vehicle (blue symbols, n = 4) six times by oral gavage (P.O.) starting 24 h p.i., and every 12 h thereafter. Mock-treated mice were used as non-infected controls (black symbols, n = 3).JQuantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice 7, 14, and 21 days p.i.Data information: Data are expressed as mean ± SEM. *P-value [less than] 0.05, **P-value [less than] 0.01, ***P-value [less than] 0.001; One-way ANOVA followed by uncorrected Fisher's LSD, each comparison stands alone. Normal distribution was verified by Shapiro–Wilk test.Source data are available online for this figure.
Nirmatrelvir treatment of mice infected with unrelated viruses (i.e., vesicular stomatitis virus [VSV] and lymphocytic choriomeningitis virus [LCMV]) did not inhibit the development of antiviral adaptive immune responses, indicating that nirmatrelvir is not per se an immune suppressive drug (Appendix Fig S5).
Of note, we did not detect viral titers in the lungs of all but one nirmatrelvir-treated mouse after homologous re-challenge (Appendix Fig S6A and B). Similarly, infection of K18-hACE2 transgenic mice with the poorly pathogenic Omicron (B.1.1.529) variant (Halfmann et al, 2022) did not allow us to evaluate clinical signs of disease. We believe that the failure to observe decreased protection upon re-infection in mice treated with nirmatrelvir (despite a prominent reduction in adaptive immunity) has to do with the limitations of the experimental setup. Case in point, we infected mice with Omicron (B.1.1.529) variant, we treated or not with nirmatrelvir and we re-challenged mice with Delta (B1.1.617) variant, known to be more pathogenic and to replicate at higher levels in mice (Shuai et al, 2022). As shown in Appendix Fig S6C and D, two of six mice that were treated with nirmatrelvir during the primary infection showed higher viral RNA in the lungs upon Delta re-challenge. Future studies with different SARS-CoV-2 variants and/or doses should assess the impact of the reduced adaptive immunity caused by nirmatrelvir treatment on viral load and the severity of disease progression upon re-challenge.
In summary, our results indicate that nirmatrelvir treatment negatively impacts the development of adaptive immune response to SARS-CoV-2 in mice. Although the mechanistic bases behind this observation were not addressed in this study, it is conceivable that this is due to insufficient antigen exposure (quantity and/or duration) of naïve B and T cells. 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 (Mercado et al, 2000; Corbin & Harty, 2004). 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). In addition, 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). Thus, the effect of antiviral therapy on adaptive immunity probably depends on the impact of such treatment on several factors including not only pathogen replication but also duration of antigen expression and presentation, activation of innate immunity, etc.
While the clinical data continue to support nirmatrelvir treatment for the prevention of severe COVID-19 in high-risk individuals (Hammond et al, 2022), the data reported here draw attention to a potential negative impact of this therapy. Whether this effect is an exclusive feature of nirmatrelvir or whether forthcoming antivirals acting on SARS-CoV-2 would have similar effects should be addressed in future studies. Although mice do not reproduce the viral rebound observed in some patients treated with nirmatrelvir, we believe that our results might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals and should inform clinical and public health policies.
Materials and Methods VirusesThe SARS-CoV-2 isolates were propagated in Vero E6-hTMPRSS2 cells. Briefly, 3 × 106 Vero E6-hTMPRSS2 cells were plated into T75 flask in DMEM 2% FBS. After 24 h, cells were inoculated with 0.001 or 0.01 MOI of SARS-CoV-2 D614G (hCoV-19/Italy/LOM-UniMI-vir1/2020; EPI_ISL_58405), SARS-CoV-2 B.1.617.2 (hCoV-19/Italy/LOM-Milan-UNIMI9615/2021, EPI_ISL_3073880), SARS-CoV-2 B.1.1.529 (hCoV-19/Italy/LOM-19182/2021, EPI_ISL_10898045), SARS-CoV-2 Omicron subvariant B5 (hCoV-19/Italy/LOM-Unimi-5882/2022, EPI_ISL_14036188), and mouse-adapted SARS-CoV-2 (rSARS2-N501YMA30) (Wong et al, 2022). Supernatant was collected 48–72 h later, centrifuged for 5 min at 500 g, aliquoted, and stored at −80°C.
VSV Indiana and LCMV WE were propagated and quantified as described (Sammicheli et al, 2016; Giovanni et al, 2020).
Nirmatrelvir synthesisReagents and solvents were purchased from commercial sources and used without further purification. Reactions were carried out at room temperature (RT), unless otherwise specified. Moisture-sensitive reactions were performed under a positive pressure of dry nitrogen in oven-dried glassware. Analytical thin-layer chromatography (TLC) on silica gel 60 F254 plates (250 μm thickness) was performed to monitor the reaction progress, using UV and KMNO4 as revelation method. Analytical HPLC was performed to monitor the reaction progress and the purity of target compound. Flash chromatography on silica gel (70—230 mesh) and preparative HPLC were performed for purification. All products were characterized by their NMR and MS spectra. (ESI)-MS spectra were performed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) by infusion into the ESI source using MeOH as solvent. 1H NMR spectra were recorded in deuterated solvents at 25°C on Bruker Avance NEO 400 MHz and 700 MHz instruments. equipped with a RT-DR-BF/1H-5 mm-OZ SmartProbe. Chemical shifts (δ) are reported in part per million (ppm) downfield from tetramethylsilane, using residual solvent signal as the internal reference.
The final compound was characterized by HPLC-MS/MS, using a Dionex ULTIMATE 3000 (Thermo Fisher Scientific) HPLC module and a LTQ XL mass spectrometer with electrospray ionization in positive mode and an Ion-Trap detector. Separation was performed with a Kinetex column C18 Polar column (250 mm × 4.6 mm; particle size 5 μm, Phenomenex, Torrance, CA, USA) at 30°C, using a 17 min gradient, 5%[0.1%TFA/CH3CN]/95%[0.1%TFA/H2O] to 95%[0.1%TFA/CH3CN]. Analytical HPLC was performed on Shimatzu-1100 HPLC using a Kinetex C18 column (4.6 mm × 150 mm, 5 μm, 100 Å) with an acetonitrile (0.1% HCOOH)−water (0.1% HCOOH) custom gradient. The purity of the final compound was > 95%, as determined by HPLC (UV λ = 200 nm). Preparative HPLC was performed on Shimatzu LC-20AP using a Sunfire C18 column (19 mm × 100 mm, 5 μm, 100 Å) with an acetonitrile (0.1% HCOOH)−water (0.1% HCOOH) custom gradient.
Briefly, two “building blocks,” the acid dipeptide P3-P2 and the residue P1, were first synthesized separately and then assembled to generate the advanced intermediate 6. 6 was deprotected to obtain intermediate 7 that was converted into the final product nirmatrelvir with one pot two steps procedure by converting 7 primary amide in P1 to the nitrile, electrophilic “warhead”, and obtaining the trifluoroacetamide, as N terminal capping group.
Methyl (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3, 3-dimethylbutanoyl)-6, 6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (3). Tert-leucine-OH 2 (1 g, 4.32 mmol) was dissolved in dry CH2Cl2 (5 ml), and amine hydrochloride 1 (1.15 g, 5.62 mmol), HBTU (1.8 g, 4.75 mmol), and DIPEA (1.5 ml, 8.64 mmol) were added at 0°C under a nitrogen atmosphere. The resulting solution was kept under magnetic stirring at RT for 16 h. Then, the reaction mixture was washed with saturated aq. NaHCO3 (x1), 1 N HCl (x1), and brine (x1), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The reaction crude was purified by flash column chromatography (Hexane / EtOAc 8: 2) to obtain a colorless oil (reaction time: 16 h; yield: 1.38 g, 78%). 1H NMR (400 MHz, DMSO-d6) δ 6.73 (d, J = 9.3 Hz, 1H), 4.21 (s, 1H), 4.05 (d, J = 9.4 Hz, 1H), 3.93 (d, J = 10.4 Hz, 1H), 3.79 (dd, J = 10.3, 5.3 Hz, 1H), 3.65 (s, 3H), 1.55–1.49 (m, 1H), 1.41 (d, J = 7.5 Hz, 1H), 1.35 (s, 9H), 1.01 (s, 3H), 0.93 (s, 9H), 0.85 (s, 3H). MS (ESI) m/z calcd: [M + H]+ for C20H35N2O5+ 383.51, found [M + H]+ 383.50.
(1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3, 3-dimethylbutanoyl)-6, 6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (4). The methyl ester intermediate 3 (1.35 g 3.6 mmol) was dissolved in THF (18 ml), then 1 N aq. LiOH was added (18 mmol, 18 ml), and the reaction mixture was kept under stirring at RT for 3 h. The reaction mixture was cooled to 0°C, placed in water / ice, acidified with 1 N HCl to pH = 4, then extracted with EtOAc (x3). Then, the collected organic layers were washed with brine (x1), dried over Na2SO4, filtered and concentrated under vacuum to afford dipeptide acid 4 as white solid which was used in the following step without further purification (reaction time: 2 h, yield: 1.30 mg, 100%). 1H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 6.67 (d, J = 9.4 Hz, 1H), 4.13 (s, 1H), 4.04 (s, 1H), 3.91 (d, J = 10.4 Hz, 1H), 3.77 (dd, J = 10.2, 5.3 Hz, 1H), 1.54–1.46 (m, 1H), 1.40 (s, 1H), 1.35 (s, 9H), 1.01 (s, 3H), 0.93 (s, 9H), 0.84 (s, 3H). MS (ESI) m/z calcd: [M + H]+ for C19H33N2O5+ 369.48, found [M + H]+ 369.50.
Tert-butyl ((S)-1-((1R,2S,5S)-2-(((S)-1-amino-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamoyl)-6, 6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3, 3-dimethyl-1-oxobutan-2-yl)carbamate (6). Dipeptide acid 4 (1.22 g, 3.3 mmol) was dissolved in dry CH2Cl2 (6 ml) and the amine hydrochloride 5 (898 mg, 4.3 mmol), HBTU (1.25 g, 3.6 mmol), and DIPEA (1.4 ml, 8.25 mmol) were added at 0°C, then DMF (3 ml) was added, and the reaction mixture was kept under stirring at RT for 3 h. The reaction mixture was washed with 1 N HCl (x3), saturated aq. NaHCO3 (x3), brine (x3), dried over Na2SO4, filtered, and concentrated under vacuum. The crude was purified by flash chromatography (CHCl3/MeOH 5 to10%) to obtain the tripeptide 6 as a white solid (reaction time: 3 h, yield: 1.3 g, 75%). 1H NMR (400 MHz, DMSO-d6) δ 9.41 (d, J = 8.6 Hz, 1H), 8.29 (d, J = 8.9 Hz, 1H), 7.55 (s, 1H), 7.31 (s, 1H), 7.03 (s, 1H), 4.43 (d, J = 8.6 Hz, 1H), 4.35–4.25 (m, 2H), 3.91–3.84 (m, 1H), 3.67 (d, J = 10.4 Hz, 1H), 3.13 (t, J = 9.0 Hz, 1H), 3.06–2.97 (m, 1H), 2.45–2.34 (m, 1H), 2.14 (dt, J = 10.5, 7.4 Hz, 1H), 1.97–1.86 (m, 1H), 1.70–1.57 (m, 1H), 1.54–1.45 (m, 2H), 1.38 (d, J = 7.7 Hz, 1H), 1.10 (s, 3H), 1.05 (s, 9H), 0.98 (s, 9H), and 0.84 (s, 3H). MS (ESI) m/z calcd: [M + H]+ for C26H44N5O6+ 522.67, found [M + H]+ 522.70.
(1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3, 3-dimethyl-2-(2, 2, 2-trifluoroacetamido)butanoyl)-6, 6-dimethyl-3 azabicyclo[3.1.0]hexane-2-carboxamide (nirmatrelvir). Tripeptide intermediate 6 (300 mg, 0.57 mmol) was dissolved in CH2Cl2 (3 ml), the solution was cooled to 0°C, 4 N HCl d in 1,4-dioxane (1.5 ml, 5.7 mmol), and the reaction mixture was stirred at RT for 2 h. Then, the solvent mixture was evaporated in vacuo and the crude was treated with hexane to obtain the desired compound 7 as HCl salt white solid, which was used without further purification in the following step (reaction time: 2 h, yield: 261 mg, 100%). Intermediate 7 (230 mg, 0.5 mmol) was suspended in dry CH2Cl2 (2 ml), under an N2 atmosphere, and dry pyridine (0.10 ml, 1.43 mmol) was added. After 30 min, the resulting mixture was cooled to 0°C, TFFA (0.08 ml, 0.57 mmol) was added, and the reaction mixture was kept under stirring at RT for 2 h. The complete conversion of the intermediate 7 was monitored by TLC, then anhydrous pyridine (0.18 ml, 2.28 mmol) was added, and the mixture was cooled at −5°C. After 5 min, TFFA (0.16 ml, 1.14 mmol) was added and the reaction was kept under stirring at RT for 18 h. The solvent was removed under vacuum, the resulting crude was diluted with EtOAc, and the organic phase was washed with 0.5 N HCl (x3), saturated aq. NaHCO3 (x1), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude was purified by preparative HPLC (Shimadzu LC-20AP; column: Sunfire, 5 μm, C18, 100 Å, 19 × 100 mm, C18 with TMS end capping; mobile phase gradient: 30–75, 20 min [H2O 0.1% HCOOH, MeCN 0.1% HCOOH]; time course: 20 min; tR = 7.8 min) to afford the target nirmatrelvir (yield: 115 mg, 40%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.43 (d, J = 8.4 Hz, 1H), 9.03 (d, J = 8.6 Hz, 1H), 7.68 (s, 1H), 4.97 (ddd, J = 10.9, 8.6, 5.1 Hz, 1H), 4.41 (d, J = 8.4 Hz, 1H), 4.15 (s, 1H), 3.91 (dd, J = 10.4, 5.5 Hz, 1H), 3.69 (d, J = 10.4 Hz, 1H), 3.17–3.11 (m, 1H), 3.04 (td, J = 9.4, 7.1 Hz, 1H), 2.40 (tdd, J = 10.4, 8.4, 4.4 Hz, 1H), 2.14 (ddd, J = 13.4, 10.9, 4.4 Hz, 1H), 2.11–2.03 (m, 1H), 1.76–1.65 (m, 2H), 1.57 (dd, J = 7.6, 5.5 Hz, 1H), 1.32 (d, J = 7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), and 0.85 (s, 3H). MS (ESI) m/z calcd: [M + H]+ for C23H33F3N5O4+ 500.53, found [M + H]+ 500.40.
The image of 1H NMR and MS spectra of nirmatrelvir are reported in Appendix Figs S7 and S8.
Biochemical assayMpro SARS-CoV-2 was expressed in E. coli cells BL21 (DE3) and purified as described (Costanzi et al, 2021; Kuzikov et al, 2021). Briefly, the protein was purified in two steps using a Ni-Sepharose column and by HiTrap Q HP column and the fractions containing the Mpro SARS-2 were pooled and concentrated using Amicon Ultra 15 centrifugal filters, at 4,000 g, at 4°C, in a buffer exchange (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 7.8). Protein purity was verified by SDS–PAGE analysis and the proteins were stored at −80°C.
The Mpro SARS-CoV-2 biochemical assays was performed in 386 wells plate in 20 μl of assay buffer containing diluted protein, 20 mM Tris (pH 7.3), 100 mM NaCl, and 1 mM EDTA, with the addition of 5 mM TCEP, 0.1% BSA. In Fig 1B, the protein was preincubated for 30 min at 37°C with different concentrations of nirmatrelvir or GC376, a commercially available broad-spectrum Mpro inhibitor (17–20). The substrate DABCYL-KTSAVLQ↓SGFRKM-EDANS (Bachem) was added, and the generation of the fluorescent product was monitored after 15 min of incubation (Ex 340 nm, Em 490 nm). In Appendix Fig S1A, the protein and the compound were not preincubated and the enzymatic reaction was immediately initiated with the addition of the substrate in the assay buffer, as described (Owen et al, 2021). The reaction was allowed to progress for 60 min at 23°C and then monitored (Ex 340 nm, Em 490 nm). Dose–response curve were generated by non-linear regression curve fitting with GraphPad Prism to calculate IC50.
For in vitro antiviral assay, HEK293T-hACE were plated in 96-well plates at 5,000 cells/well in complete DMEM plus 2% FBS. After 24 h, cells were treated with seven concentrations of fivefold serially diluted nirmatrelvir and infected at 0.1 MOI of SARS-CoV-2 virus. DMSO was used as vehicle for compound serial dilution and not treated control (final concentration 0.25%). Not infected condition was inserted as negative control of infection. Each condition was assayed in three replicates. Antiviral activity was evaluated by qPCR quantification of secreted SARS-CoV-2 RNA and/or by cytopathic effect protection assay (CPE) after 72 h of incubation at 37°C under 5% CO2.
For the quantification of SARS-CoV-2 RNA by qPCR, 10 μl of cell supernatants were subjected to direct lysis with the addition of 10 μl ViRNAex solution (Cabru) and heated at 70°C for 15 min. After addition of distilled water (1:1), samples were used as template for PCR amplification using TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific) and specific SARS-CoV-2 primers/probe (2019-nCoV RUO Integrated DNA Technologies). Obtained Ct were normalized to untreated infected wells, and dose–response curve were generated by non-linear regression curve fitting with GraphPad Prism to calculate the concentration that inhibits 50% of viral replication (IC50).
For CPE assays, CellTiter-Glo® Luminescent Cell Viability Assay (Promega), was used. Relative luciferase units (RLUs) were normalized to infected or uninfected controls in order to obtain the percentage of inhibition of cytopathic effect using the following formula: % CPE inhibition = 100*(Test Cmpd − Avg. Virus)/(Avg. Cells − Avg. Virus), where Avg. virus is the RLU average obtained from infected and not treated wells, while Avg. cells is the RLU average obtained from not infected and not treated wells. Dose–response curve was generated by non-linear regression curve fitting with GraphPad Prism to calculate IC50.
MiceB6.Cg-Tg(K18-ACE2)2Prlmn/J mice (referred to in the text as K18-hACE2) were purchased from The Jackson Laboratory. C57BL/6 mice were purchased from Charles River. Mice were housed under specific pathogen-free conditions, and heterozygous mice were used at 8–10 weeks of age. All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces.
Mouse infectionInfection of K18-hACE2 transgenic mice with aerosolized SARS-CoV-2 was performed as described (Fumagalli et al, 2021). Briefly, non-anesthetized K18-hACE2 mice were placed in a nose-only Allay restrainer on the inhalation chamber (DSI Buxco respiratory solutions, DSI). To reach a target accumulated inhaled aerosol (also known as delivered dose) of 2 × 105 TCID50 mice were exposed to aerosolized SARS-CoV-2 B.1.1.529 or B.1.617.2 for 30–60 min (depending on the total volume of diluted virus and on the number of mice simultaneously exposed). In selected experiments, mice were exposed to a target accumulated inhaled aerosol of 1 × 106 TCID50. In Fig 3I and J, and Appendix Fig S4, C57BL/6 mice were exposed to a target dose of 1 × 105 TCID50 of the mouse-adapted SARS-CoV-2 strain (rSARS-N501YMA30). Primary inflows and pressure were controlled and set to 0.5 l/min/port and −0.5 cm H2O, respectively. As control, K18-hACE2 mice or C57BL/6 mice received the same volume of aerosolized PBS (125 μl per mouse). Infected mice were monitored daily to record body weight, and clinical and respiratory parameters.
C57BL/6 WT mice were infected intravenously with 1 × 106 plaque-forming units (pfu) of VSV Indiana and 2.5 × 106 pfu of LCMV WE.
K18-hACE2 mice were treated by oral gavage with nirmatrelvir at 150 mg/kg or vehicle (0.5% Methylcellulose (Methocel A4M, Sigma #94378) and 2% Tween80 (Sigma #8170611000) in purified water) for six times starting 4 h post-infection, and every 12 h thereafter. In selected experiments, K18-hACE2 mice or C57BL/6 mice were treated with nirmatrelvir starting 1 or 2 days post-infection (Fig 3).
LC–MS/MS analysisThe nirmatrelvir stock solutions were prepared in DMSO at 1 mg/ml and further diluted to obtain a working solution (WS) at 20 μg/ml. The drug JWH-250 was used as internal standard. The internal standard working solutions (IS-WS) was prepared at 20 ng/ml in methanol:acetonitrile (50:50, v/v) acidified with 0.1% formic acid.
Plasma of mice were collected, centrifuged at 10,621 g for 10 min, and incubated at 60°C for 30 min. The mixture of 30 μl of plasma, 105 μl of IS-WS, and 15 μl of WS was vortex for 1 min and centrifuged at 12,500 g for 10 min at 4°C. The supernatant was collected and 100 μl were injected into the liquid chromatography tandem mass spectrometry (LC–MS/MS) system. The HPLC equipment consisted of an LC AC System from AB Sciex (Toronto, ON, Canada). A Triple Quadrupole Mass Spectrometer (API 2000) from AB-Sciex (Toronto, ON, Canada) was used for detection. The analytes were separated using an Acquity UPLC BEH C18 1.7 μm Column (2.1 × 50 mm ID) from Waters. The mobile phases were (B) MeOH containing 0.2% formic acid and (A) water containing 0.1% formic acid, at a flow rate of 0.4 ml/min, and were entirely transferred into the mass spectrometer source. The gradient elution was as follows: increase in the organic phase from 10 to 100% in 2 min and after 1.5 min of 100% B, the column was led to the original conditions; equilibration of the column was achieved in 2 min. Both analytes were detected in positive ionization with a capillary voltage of 4,500 V, nebulizer gas (air) at 45 psi, turbo gas (nitrogen) at 70 psi, and 450°C. The other ion source parameters were set as follows: curtain gas (CUR) 25 psi; collision gas (CAD) 6 psi; declustering potential 80 V; and entrance potential 8 V. Instrument conditions optimization was performed by direct infusion and manual tuning. Data collection and elaboration were performed by means of Analyst 1.4 software (AB- Sciex). The quantitative data were acquired using multi reaction monitoring (MRM) mode. Two MRM transitions (precursor ion > fragment ion) were selected for the analytes. The parameters used for each analyte are listed in the Appendix Table S1.
The analytical method was validated according to FDA guidelines for bioanalytical method validation. Linearity, precision, accuracy, limits of detection (LODs), and limits of quantification (LOQ) were evaluated. Calibration standard solutions were prepared in blank plasma by spiking 15 μl of a standard mixture at appropriate concentration to 30 μl of plasma and by adding 105 μl of methanol:acetonitrile (50:50, v/v). Calibrators were then treated similarly to the animal samples. The calibration range was 2–750 ng/ml and the calibrators were prepared at nine level of concentration. Precision, recovery, and accuracy were evaluated at three level of concentrations (25, 100, and 750 ng/ml) and resulted within the acceptable limits. LOD was defined as the lowest concentration with a signal-to-noise (S/N) ratio greater than 3. LOQ was defined as the concentration at which both precision (RSD %) and accuracy were less than 20%. LOQ resulted to be 2 ng/ml, while LOD was 1 ng/ml for both analytes.
Tissue homogenate and viral titersTissues homogenates were prepared by homogenizing perfused lungs using gentleMACS Octo Dissociator (Miltenyi BioTec, #130-096-427) in M tubes (#130-093-335) containing 1 ml of DMEM 0% FBS. Samples were homogenized for three times with program m_Lung_01_02 (34 s, 164 rpm). The homogenates were centrifuged at 2,000 g for 5 min at 4°C. The supernatant was collected and stored at −80°C for viral isolation and viral load detection. Viral titer was calculated by 50% tissue culture infectious dose (TCID50). Briefly, Vero E6 cells were seeded at a density of 1.5 × 104 cells per well in flat-bottom 96-well tissue culture plates. The following day, 10-fold dilutions of the homogenized tissue were applied to confluent cells and incubated 1 h at 37°C. Then, cells were washed with phosphate-buffered saline (PBS) and incubated for 72 h at 37°C in DMEM 2% FBS. Cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.05% (wt/vol) crystal violet in 20% ethanol. Limit of detection (LOD) was defined as the lowest concentration whereby the virus, used as positive control, has killing capacity of cells.
RNA extraction and qPCRTissues homogenates were prepared by homogenizing perfused lung and nasal turbinates using gentleMACS dissociator (Miltenyi BioTec, #130-096-427) with program RNA_02 in M tubes (#130-096-335) in 1 ml of Trizol (Invitrogen, #15596018). The homogenates were centrifuged at 2,000 g for 1 min at 4°C and the supernatant was collected. RNA extraction was performed by combining phenol/guanidine-based lysis with silica membrane-based purification. Briefly, 100 μl of chloroform was added to 500 μl of homogenized sample and total RNA was extracted using ReliaPrep™ RNA Tissue Miniprep column (Promega, Cat #Z6111). Total RNA was isolated according to the manufacturer's instructions. qPCR was performed using TaqMan Fast virus 1 Step PCR Master Mix (Life Technologies #4444434), standard curve was drawn with 2019_nCOV_N Positive control (IDT#10006625), and primers used are as follows: 2019-nCoV_N1- Forward Primer (5′-GAC CCC AAA ATC AGC GAA AT-3′), 2019-nCoV_N1- Reverse Primer (5′-TCT GGT TAC TGC CAG TTG AAT CTG-3′), and 2019-nCoV_N1-Probe (5′-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3′) (Centers for Disease Control and Prevention (CDC) Atlanta, GA 30333). All experiments were performed in duplicate.
ELISAIndividual sera were titrated in parallel for the presence of SARS-CoV-2 S1 RBD-specific antibody by end-point ELISA. The ELISA plates were functionalized by coating with recombinant Sars-CoV-2 S1 subunit protein (RayBiotech, #230-30162) at a concentration of 2 μg/ml and incubated overnight (O/N) at 4°C. Subsequently, the plates were blocked with 3% fat-free milk and 0.05% Tween20 in PBS for 1 h at RT. The sera were then added at a dilution of 1/20 (sera from day 7) or 1/500 (sera from day 14, 21, and 28) and diluted 1:10 up to 1/1,280 or 1/32,000, respectively, in duplicate, and the plates were incubated for 2 h at RT. After five washes with 0.05% Tween20 in PBS, the secondary anti-murine IgG conjugated with horseradish peroxidase (HRP, PerkinElmer, #NEF822001EA) (1:2,000) was added and the plates were incubated for 1 h at RT. After washing, the binding of the secondary was detected by adding the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, BD Biosciences). The reaction was blocked with 0.5 M H2SO4 and the absorbance at 450 nm and reference 630 nm was measured.
Individual sera were titrated in parallel for the presence of VSV-specific IgG by end-point ELISA. Neutralizing dose 50 were measured as described (Sammicheli et al, 2016).
SARS-CoV-2 pseudovirus neutralization assaySARS-CoV-2 pseudovirus neutralization assay was performed as previously described (Conforti et al, 2022). Briefly, lentiviral vector containing luciferase reporter were pseudotyped with B.1.1.529 SARS-CoV-2 spike protein and used for neutralization assay. HEK293T-hACE2 receptor were plated in 96-well plates and transduced with 0.05 MOI of SARS-CoV-2 pseudovirus that were subjected to 1 h at 37°C of preincubation with threefold serially diluted mice plasma. After 24 h of incubation, pseudoparticle cell transduction was measured by luciferase assay using Bright-Glo™ Luciferase System (Promega), and dose–response curves were generated by non-linear regression curve fitting to calculate Neutralization dose 50 (ND50).
Cell isolation and flow cytometryMice were euthanized by cervical dislocation. At the time of autopsy, mice were perfused through the right ventricle with PBS. Nasal turbinates were removed from the nose cavity. Lung tissue was digested in RPMI 1640 containing 3.2 mg/ml Collagenase IV (Sigma, #C5138) and 25 U/ml DNAse I (Sigma, #D4263) for 30 min at 37°C. Homogenized lungs were passed through 70 μm nylon meshes to obtain a single cell suspension. Cells were resuspended in 36% percoll solution (Sigma, #P4937) and centrifuged for 20 min at 653 g (light acceleration and low brake). The remaining red blood cells were removed with ACK lysis. Peripheral blood was collected in PBS 0.5 mM EDTA and lysed two times with ACK. Single-cell suspensions of spleens were generated as described (Sammicheli et al, 2016).
For analysis of ex-vivo intracellular cytokine production, 1 mg/ml of brefeldin A (Sigma, #B7651) was included in the digestion buffer. All flow cytometry stainings of surface-expressed and intracellular molecules were performed as described (Bénéchet et al, 2019; Fumagalli et al, 2021, 2022; Simone et al, 2021). Briefly, cells were stimulated for 4 h at 37°C in the presence of brefeldin A, monensin (life technologies, #00-4505-51), and a pool of overlapping peptides (1 μg/ml per peptide), including MHC class I- and MHC class II-restricted peptides (9–22 amino acids) covering the S, S1, S+, M, and N protein of SARS-CoV-2 (Miltenyi, #130-126-700; #130-127-041; #130-127-311; #130-126-702, #130-126-698; Silva-Cayetano et al, 2021). In selected experiments (Appendix Fig S5), cells were stimulated with GP61 and GP33 peptides (2 μg/ml per peptide) (Iannacone et al, 2008). As positive control for IFN-γ and TNF-α production, cells were stimulated with PMA (Invitrogen, # 356150050) and Ionomycin (Invitrogen, #I24222). Cell viability was assessed by staining with Viobility™ 405/520 fixable dye (Miltenyi, Cat #130-109-814). In Fig 2H, biotinylated-RBD (26 KDa, kindly provided by L. Aurisicchio from Takis Biotech) were mixed with Alexa Fluor (AF)-647 or 488-fluorescent streptavidins (53 kDa) in a molar ratio of 4:1, respectively, to obtain fluorescent RBD tetramers at 2 μg/ml. RBD-specific B cells were labeled prior to the surface staining for 30 min at 4°C. Antibodies (Abs) are indicated in Appendix Table S2.
Flow cytometry analysis was performed on BD FACS Symphony A5 SORP, Cytek Aurora, and analyzed with FlowJo software (Treestar).
Confocal immunofluorescence histologyMice were euthanized and perfused transcardially with PBS. One left lobe of the lung was collected and fixed in 4% paraformaldehyde for 16 h, then dehydrated in 30% sucrose prior to embedding in OCT freezing media (Killik Bio-Optica #05-9801). Twenty micrometer sections were cut on a CM1520 cryostat (Leica) and adhered to Superfrost Plus slides (Thermo Scientific). Sections were permeabilized and blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and 0.5% BSA followed by staining in permeabilization buffer of Foxp3 / Transcription Factor Staining Buffer Set (eBioscence, # 00-5523-00). Slides were stained for Ki-67 (eBioscence, Clone SolA15, # 56-5698-82) and B220 (Biolegend, Clone RA3-6B2, #103228) overnight at RT. Lung sections were washed twice for 5 min and stained with DAPI (Life Technologies, #D1360) for 5 min at RT, then washed again, and mounted for imaging with FluorSaveTM Reagent (Merck Millipore, #345789). Images were acquired on an SP5 or SP8 confocal microscope with 20× objective (Leica Microsystem). To minimize fluorophore spectral spillover, the Leica sequential laser excitation and detection modality was used.
Statistical analyses and softwareDetailed information concerning the statistical methods used is provided in the figure legends. Flow data were collected using FlowJo Version 10.5.3 (Treestar). Statistical analyses were performed with GraphPad Prism software version 8 (GraphPad). Immunohistochemical imaging analyses were performed with QuPath (Quantitative Pathology & Bioimage 5 Analysis) software. N represents individual mice analyzed per experiment. Experiments were performed independently at least twice to control for experimental variation. Error bars indicate the standard error of the mean (SEM). In selected experiments (Fig 1B and C, Appendix Figs S1A and B, and S4D), error bars indicate the standard deviation (SD). Dose–response curves for IC50 values were determined by non-linear regression. We used Mann–Whitney U-tests to compare two groups with non-normally distributed continuous variables and Kruskal–Wallis non-parametric test or one-way ANOVA test to compare three or more unpaired groups. Normality of data distribution was tested with a Shapiro–Wilk normality test and normality was chosen only when normality could be confirmed for each dataset. We used two-way ANOVA followed by Sidak's multiple comparisons tests to analyze experiments with multiple groups and two independent variables. Significance is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Comparisons are not statistically significant unless indicated.
Data availabilityAll data are available in the main text or the appendix file. This study includes no data deposited in external repositories.
AcknowledgementsWe thank S. De Palma, A. Fiocchi, L. Giustini, M. Mainetti, and C. Perucchini for technical support; M. Silva and M. Tinelli for secretarial assistance; L. Aurisicchio (Takis Biotech) for providing biotinylated-RBD, and the members of the Iannacone laboratory for helpful discussions. Flow cytometry was carried out at FRACTAL, a flow cytometry resource and advanced cytometry technical applications laboratory established by the San Raffaele Scientific Institute. Confocal immunofluorescence histology was carried out at Alembic, an advanced microscopy laboratory established by the San Raffaele Scientific Institute and the Vita-Salute San Raffaele University. We would like to acknowledge the PhD program in Basic and Applied Immunology and Oncology at Vita-Salute San Raffaele University, as DM conducted this study as partial fulfillment of their PhD in Molecular Medicine within that program. M.I. is supported by the European Research Council (ERC) Consolidator Grant 725038, ERC Proof of Concept Grant 957502, Italian Association for Cancer Research (AIRC) Grants 19891 and 22737, Italian Ministry of Health Grants RF-2018-12365801, and Funded Research Agreements from Gilead Sciences, Asher Bio, Takis Biotech, and Vir Biotechnology. L.G.G. is supported by the Italian Association for Cancer Research (AIRC) Grant 22737, Lombardy Open Innovation Grant 229452, PRIN Grant 2017MPCWPY from the Italian Ministry of Education, University and Research, Funded Research Agreements from Gilead Sciences, Avalia Therapeutics, and CNCCS SCARL and a donation from FONDAZIONE SAME for COVID-19-related research. V.F. is supported by a donation from FONDAZIONE PROSSIMO MIO and from AIRC Fellowship for Italy (ID 26813-2021). This research was funded, in part, by EU funding within the NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT). Open access funding provided by BIBLIOSAN.
Author contributionsValeria Fumagalli: Data curation; formal analysis; investigation; visualization; methodology; writing – review and editing. Pietro Di Lucia: Investigation. Micol Ravà: Investigation. Davide Marotta: Investigation. Elisa Bono: Investigation. Stefano Grassi: Resources. Lorena Donnici: Investigation. Rolando Cannalire: Investigation. Irina Stefanelli: Investigation. Anastasia Ferraro: Investigation. Francesca Esposito: Investigation. Elena Pariani: Resources. Donato Inverso: Investigation. Camilla Montesano: Investigation. Serena Delbue: Resources. Stanley Perlman: Resources. Enzo Tramontano: Resources; formal analysis. Raffaele De Francesco: Resources; formal analysis; writing – review and editing. Vincenzo Summa: Resources; formal analysis; writing – review and editing. Luca G Guidotti: Conceptualization; funding acquisition; writing – review and editing. Matteo Iannacone: Conceptualization; supervision; funding acquisition; writing – original draft; project administration; writing – review and editing.
Disclosure and competing interests statementMI participates in advisory boards/consultancies for Gilead Sciences, Third Rock Ventures, Antios Therapeutics, Asher Bio, GentiBio, Clexio Biosciences, Sybilla, BlueJay Therapeutics. LGG is a member of the board of directors at Genenta Science and participates in advisory boards/consultancies for Antios Therapeutics, Chroma Medicine, Ananda Immunotherapies, and Gilead Sciences. RDF is a consultant for Moderna and a member of the board of directors of T-One Therapeutics.
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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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1 Division of Immunology, Transplantation, and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy
2 Division of Immunology, Transplantation, and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy
3 Pathology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
4 INGM - Istituto Nazionale di Genetica Molecolare “Romeo ed Erica Invernizzi”, Milan, Italy
5 Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Naples, Italy
6 Dipartimento di Scienze della Vita e dell'Ambiente, Cittadella Universitaria di Monserrato, Cagliari, Italy
7 Department of Biomedical Sciences for Health, University of Milan, Milan, Italy
8 Department of Chemistry, La Sapienza University, Rome, Italy
9 Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy
10 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA; Department of Pediatrics, University of Iowa, Iowa City, IA, USA
11 INGM - Istituto Nazionale di Genetica Molecolare “Romeo ed Erica Invernizzi”, Milan, Italy; Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy
12 Division of Immunology, Transplantation, and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy; Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy