Exploratory analyses of Immunologic Features in a Randomized, Placebo-Controlled Trial of Nirmatrelvir/Ritonavir for Long COVID

Bornali Bhattacharjee; Mitsuaki Sawano; William B. Hooper; Kexin Wang; Alexandra Tabachnikova; Valter Silva Monteiro; Peiwen Lu; Pavlina Baevova; Gisele C. Rodrigues; Victoria L. Fisher; Cesar Caraballo; Rohan Khera; Shu-Xia Li; Jeph Herrin; Dany Christian; Andreas Coppi; Frederick Warner; Julie Holub; Yashira Henriquez; Maria A. Johnson; Theresa B. Goddard; Erica Rocco; Amy C. Hummel; Mohammad AL Mouslmani; Kevin D. Carr; Lawrence Charnas; Magdia De Jesus; Dale Nepert; Paula Abreu; Frank W. Ziegler; John Spertus; Leying Guan; Harlan Krumholz; Akiko Iwasaki

Bornali Bhattacharjee, Mitsuaki Sawano, William B Hooper, Kexin Wang, Alexandra Tabachnikova, Valter Silva Monteiro, Peiwen Lu, Pavlina Baevova, Gisele C Rodrigues, Victoria L Fisher, Cesar Caraballo, Rohan Khera, Shu-Xia Li, Jeph Herrin, Dany Christian, Andreas Coppi, Frederick Warner, Julie Holub, Yashira Henriquez, Maria A Johnson, Theresa B Goddard, Erica Rocco, Amy C Hummel, Mohammad Al Mouslmani, Kevin D Carr, Lawrence Charnas, Magdia De Jesus, Dale Nepert, Paula Abreu, Frank W Ziegler III, John A Spertus, Leying Guan, Harlan M Krumholz, Akiko Iwasaki

doi:10.64898/2026.02.24.26347001

This exploratory analysis of PAX LC, a Phase 2, 1:1 randomized, double-blind, superiority, placebo-controlled trial examined whether treatment with nirmatrelvir/ritonavir (NMV/r) versus placebo/ritonavir (PBO/r) in individuals with Long COVID could reveal immune features associated with symptom improvement. Eightytwo participants (n=45 PBO/r; n=37 NMV/r) provided blood samples at baseline (Day 0) and post-treatment (Day 28). Baseline demographic and immunological phenotypes were similar in the two groups. No significant differences were observed in major immune cell populations or organ function markers between NMV/r vs. PBO/r groups, or before vs. after the treatment. Modest hematologic changes were noted in the NMV/r arm. SARS-CoV-2-specific IgG levels remained constant, with changes in total immunoglobulin subtypes and isotypes in both arms. Both arms showed similar shifts in cytokine levels. Notably, the levels of S1 and Spike proteins in circulation remained unchanged post-treatment. Regardless of the treatment arm, participants with selfreported symptom improvement showed reductions in the level of the inflammatory chemokine RANTES. Taken together, the findings of this study demonstrate limited virological and immunological changes in response to nirmatrelvir, contributing insights into the reason for the lack of benefit of the 15-day NMV/r treatment in Long COVID.

Materials and Methods Study design and patient characteristics The PAX LC study was a decentralized, phase 2, randomized, double-blind, placebocontrolled clinical to investigate the efficacy and safety of a 15-day regimen of orally administered nirmatrelvir/ritonavir compared with placebo/ritonavir in participants with Long COVID. The details of the study has been published elsewhere 8 . Briefly, the key inclusion criteria for participants were: being 18 Biospecimen collection Whole blood samples were collected in lithium-heparin-coated (BD 367880, BD Biosciences) and sodium-EDTA coated vacutainers (BD 367856, BD Biosciences) from participants' homes, by ExamOne phlebotomists (Quest Diagnostics) or at the Yale clinic, New Haven, CT. After collecting, biospecimens were either shipped overnight at regulated temperatures or handed over locally to researchers at Yale University in New Haven, CT. Collection tubes were de-identified upon receipt according to protocol and study identifiers were provided. Samples were processed within 48 hours of collection. Biorender 41 was used to create a graphical schematic of the CONSORT flow chart, study design, cohorts and assays. Linear Peptide Profiling SERA serum screening A detailed description of the SERA assay has been published 22 . For this study, plasma was incubated with a fully random 12-mer bacterial display peptide library (1 × 10 10 diversity, 10-fold oversampled) at a 1:25 dilution in a 96-well, deep well plate..

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DOI record: { "DOI": "10.64898/2026.02.24.26347001", "URL": "http://dx.doi.org/10.64898/2026.02.24.26347001", "abstract": "This exploratory analysis of PAX LC, a Phase 2, 1:1 randomized, double-blind, superiority, placebo-controlled trial examined whether treatment with nirmatrelvir/ritonavir (NMV/r) versus placebo/ritonavir (PBO/r) in individuals with Long COVID could reveal immune features associated with symptom improvement. Eighty-two participants (n=45 PBO/r; n=37 NMV/r) provided blood samples at baseline (Day 0) and post-treatment (Day 28). Baseline demographic and immunological phenotypes were similar in the two groups. No significant differences were observed in major immune cell populations or organ function markers between NMV/r vs. PBO/r groups, or before vs. after the treatment. Modest hematologic changes were noted in the NMV/r arm. SARS-CoV-2-specific IgG levels remained constant, with changes in total immunoglobulin subtypes and isotypes in both arms. 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