CROI 2024 Abstract eBook

Abstract eBook

Oral Abstracts

136

Molnupiravir Does Not Increase 3CLpro Resistance Mutations When Co-Administered With Nirmatrelvir/r Shuntai Zhou 1 , Nathan Long 1 , Kyle Rosenke 2 , Michael A. Jarvis 2 , Heinz Feldmann 2 , Ronald Swanstrom 1 1 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2 National Institute of Allergy and Infectious Diseases, Hamilton, MT, USA Background: Current treatments for SARS-CoV-2 rely on direct-acting antivirals including the 3CLpro inhibitor nirmatrelvir/r (NMV/r) and the mutagenic nucleoside analog molnupiravir (MOV). There have been concerns regarding the use of MOV including the generation of new viral variants. In a previous study, we compared the antiviral effect of MOV or NMV/r alone, or the co-administration of both in a SARS-CoV-2 macaque model. We showed an additive effect of the two antivirals on several markers of disease. In this follow- up study, we investigated the mutation profiles of SARS-CoV-2 in samples collected in the previous study. Methods: Twenty macaques received the oral treatment of MOV, NMV/r, combination therapy or vehicle control (5 in each group) starting 12 hours after exposure to SARS-CoV-2, and they were followed for 4 days before necropsy. Viral RNA was extracted from lung tissue samples at necropsy. Primer ID sequencing approach was used to sequence nsp5 (3CLpro), a portion nsp12 (RdRp) and the S gene receptor binding domain (S-RBD). Substitutions with a FDR-adjusted p value of less than 0.05 were considered as true mutations. This work was partially funded by the Intramural Research Program, NIAID. Results: In the MOV group, the overall substitution rate was approx. 0.05%, significantly higher than other groups. The mutation profile was driven by the increase of C-to-U and G-to-A mutations. The overall substitution rates were significantly increased in the MOV+NMV/r group compared with NMV/r or vehicle groups, but lower than MOV group (Fig 1A). We further studied the impact of MOV on the potential development of resistance mutations on 3CLpro against NMV. We did not observe any mutation hotspots in this region beyond those mediated by MOV. Mutations identified in the MOV group were largely distinct in frequency from those in the combination group. We assessed the amino acid codon changes at four positions (L50F, E166A, E166V, and L167F) that have been reported to contribute to NMV resistance. We did observe an increase in the abundance of L50F in the MOV group but it was not further enriched in the combination group (Fig 1B). Conclusion: Our results suggest that co-administration of NMV/r lowered the magnitude of the mutagenetic effect of MOV against SARS-CoV-2, likely due to the additive effect of the combination therapy on reduced rounds of viral replication. There is no evidence that combination therapy potentiated selection for NMV resistance mutations during 4 days of treatment. The figure, table, or graphic for this abstract has been removed. Mini-Lecture: Progress in Understanding the Mechanisms of Long COVID Annukka A Antar The Johns Hopkins University, Baltimore, MD, USA Background: Millions of people across the globe have experienced new or persistent symptoms for 3 or more months following COVID-19, a syndrome termed long COVID. The US Census estimates that over 15% of US adults have ever had long COVID. Investigations are underway to understand the biologic mechanisms of long COVID and develop effective therapeutics. This mini lecture will highlight the progress made in the past year on understanding the pathogenesis of long COVID. Multimodal Assessment of Antigen Persistence in the Post-Acute Phase of SARS-CoV-2 Infection Michael J Peluso 1 , Sarah Goldberg 1 , Zoe Swank 2 , Brian H. Lafranchi 1 , Scott Lu 1 , Thomas Dalhuisen 1 , Badri Viswanathan 1 , Ma Somsouk 1 , J. D. Kelly 1 , Steven G. Deeks 1 , Zoltan Laszik 1 , David Walt 2 , Jeffrey Martin 1 , Timothy J. Henrich 1 , for the LIINC Study Team 1 University of California San Francisco, San Francisco, CA, USA, 2 Brigham and Women's Hospital, Boston, MA, USA Background: Although RNA viruses like SARS-CoV-2 are considered transient, viral components can persist beyond the acute phase due to various virologic and immunologic factors. Recent studies have suggested that SARS-CoV 2 antigens may persist following COVID-19 but were limited by a lack of comparison to true negative control samples. Methods: We assessed viral persistence in two ways: (1) Single molecule array (Simoa) assays for SARS-CoV-2 spike, S1, and nucleocapsid antigen in plasma

from 171 individuals in the post-acute phase of SARS-CoV-2 infection and 250 pre-pandemic control samples, and (2) RNAscope assessing SARS-CoV-2 spike RNA in situ in rectal tissue obtained via flexible sigmoidoscopy in 5 individuals between 90 and 676 days post-COVID (without reinfection), with H&E and immunohistochemical visualization of CD3 and CD68 to localize viral RNA signals within tissue regions and immune cell types. Results: In plasma, compared to the proportion of antigen positivity in pre-pandemic controls (2.0%), detection of any SARS-CoV-2 antigen was more frequent across all post-acute COVID-19 time periods (3-6 months: 12.6%, p<0.001; 6-10 months, 10.7%, p=0.0002; 10-14 months, 7.5%, p=0.017; (a)). These differences were driven by spike for up to 14 months and nucleocapsid in the first 6 months after infection. Hospitalization for acute COVID-19 was associated with detectable antigen in the post-acute phase (OR 2.27, p=0.054) and strongly associated with detectable N antigen (OR 11.82, p=0.001). In gut, RNAscope revealed readily detectable SARS-CoV-2 RNA in multiple cells from all rectal tissue regions surveyed from 4/5 individuals (b), except for one who had rare RNA+ cells detected in 1/3 regions. Nearly all RNA+ cells were detected in the lamina propria, without an epithelial signal. A small percentage of RNA+ cells expressed CD68, a monocyte marker, but many RNA+ cells did not express CD68 and none expressed CD3. In 3/4 samples with readily detected RNA, the signal was associated with macrophage-dense areas. Conclusion: Our findings provide strong evidence that SARS-CoV-2 antigens can persist beyond the period of acute illness. The observation that 7-13% of plasma samples for over a year following initial SARS-CoV-2 infection contain detectable viral antigens, which are potentially immunogenic, has significant implications given the sheer number of people infected with SARS-CoV-2 to date. Work to determine if persistent antigen contributes to post-acute sequelae such as Long COVID is needed.

Oral Abstracts

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HIV-1 Transcription Start Sites Usage and Its Impact on Unspliced RNA Functions In Vivo Saiful Islam 1 , Zetao Cheng 1 , Olga Nikolaitchik 1 , Robert Gorelick 2 , Vinay K. Pathak 1 , Frank Maldarelli 1 , Wei-Shau Hu 1 1 National Cancer Institute, Frederick, MD, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA Background: HIV-1 unspliced RNA plays two crucial roles in viral replication: it is packaged into particles to serve as viral genome, and it is translated to generate Gag/Gag-Pol polyproteins required for virus assembly and replication. Studies using cell culture systems demonstrated that HIV-1 transcription can initiate at three conserved consecutive guanosines located at the junction of U3 and R regions, producing RNAs containing three, two, or one guanosine at the 5' end, referred to as 3G, 2G, and 1G RNA, respectively. Furthermore, 1G RNA selectively packaged over 3G RNA into viral particles to serve as virion genome, suggesting these almost identical HIV-1 RNA species differ functionally. To investigate whether HIV-1 uses multiple transcription start sites and preferentially packages a specific RNA species in vivo, we examined HIV-1 unspliced RNA in paired PBMC and plasma samples collected from infected individuals.

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CROI 2024