Proof of stability of a RSV Controlled Human Infection Model challenge agent
Authors: Verstraelen Sandra , Roymans Dirk , Jacobs An , Hollanders Karen , Remy Sylvie , Jochmans Dirk , Klein Jelle , Grauwet Tini 
Published: February 2024

Abstract
In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), a RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies. It is known that the stability of RSV can be impacted by multiple environmental factors like temperature or pH. Therefore, we wanted to assess the stability of the viral titer of RSV-NICA after five years of controlled storage and determine the homogeneity of the viral titers across the different vials of a GMP-qualified batch of RSV-NICA. In addition, we assessed the potential of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the primary target cells of RSV, and we tested the impact of single and repeated freeze-thaw cycles on the infectious viral titer of the challenge agent. The aliquoted RSV-NICA virus stock was analyzed with standard virological and molecular methods to obtain information on the titer and homogeneity of the viral titer present in 24 representative vials of the stock. Our results demonstrate that in a period of five years of cryo-storage, the infectious viral titer in 75% of the vials tested displayed a similar average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). Significant reduction down to an undetectable level of infectious virus was found in the other vials. RSV-NICA was shown to effectively infect differentiated human airway epithelial cells, and active virus replication was detected in these cells by demonstrating increasing RSV RNA titers over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating frequency in conjunction with an increase in viral RNA titers while no clear impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these pooled vials, showed no deterioration of the infectious titer. Moreover, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) in combination with subjecting the vials to three repeated freeze-thaw cycles, again did not result in a decrease of the infectious titers in the tested vials. Collectively, our data indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may have an impact on the infectious viral titer of the virus, decreasing homogeneity of this titer throughout the challenge stock. However, we also demonstrate that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration is a viable method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during RSV CHIM. RSV-NICA is a stable, adequate CHIM challenge agent that can be used in efficacy trials for RSV vaccines and antiviral entities.