Use of qualitative integrative cycler PCR (qicPCR) to identify optimal therapeutic dosing time-points in a Respiratory Syncytial Virus Human Viral Challenge Model (hVCM)
Introduction
Respiratory syncytial virus (RSV) is an enveloped, non-segmented negative-sense RNA virus in the paramyxoviridae family and is the one of the most important causes of serious lower respiratory-tract illness (LRI) worldwide (Oshansky et al., 2009). Clinical manifestations primarily occur in infants, young children, elderly, or the immunocompromised, with the greatest morbidity and mortality occurring in infants and young children with an estimated 33.8 million cases per year of RSV lower respiratory disease in children under 5 years of age (Nair et al., 2010). However, there is also a substantial morbidity and mortality amongst the elderly (Falsey et al., 2014, Matias et al., 2014). The development of novel prevention and treatment strategies should be accelerated as a priority, as no efficacious RSV treatment or vaccine is available despite decades of drug discovery research.
One of the major challenges for assessing antiviral therapeutic efficacy of new RSV therapeutics is the narrow window for antiviral intervention due to the acute nature of the RSV infection with a peak viral titer around day 3 or 4 post-infection and declining rapidly thereafter (Bagga et al., 2013). Therefore administration of anti-RSV investigational medical products (IMPs) at late stages of infection makes it difficult to accurately measure efficacy due to rapidly diminishing viral loads. An informed dosing clinical protocol is designed to ensure new antiviral therapeutic IMPs are administered immediately after RSV infection is established in a human volunteer permitting maximum IMP exposure at the time of maximal viral replication (Collins, 2013). A confounding factor in achieving this aim is the asynchronous nature for onset of RSV infection in a cohort of RSV inoculated volunteers, leading to uncertainty as to the precise start of infection in individual volunteers post-inoculation.
The integrated cycler PCR machine (Focus Diagnostics) is approved by the FDA for fast, sensitive, diagnostic testing of RSV infections in hospitals and other healthcare facilities under conditions where the infections are well-established with patients shedding high quantities of virus. However it was unclear whether it would be sufficiently sensitive to detect the low, sub-clinical RSV titres during onset of infection in asymptomatic volunteers and, secondly whether the on-board chemistry would allow use of saline nasal washes. This paper discusses the novel use of the integrated cycler assay outside of diagnostic healthcare facilities to facilitate the design of improved clinical protocols for measuring antiviral efficacy of IMPs in a clinical phase 2 setting.
Moreover, we describe the first use of qicPCR methodology to design an informed dosing clinical protocol to identify onset of RSV infection prior to a therapeutic dosing window. The success of this qicPCR approach has recently been highlighted in an informed dosing protocol designed to monitor the therapeutic efficacy for the RSV antiviral GS-5806 (DeVincenzo et al., 2014). The placebo data from 53 human volunteers from the GS-5806 study will be discussed in this paper to evaluate the effectiveness of qicPCR as a trigger for therapeutic dosing. The qicPCR assay validation data and RSV detection rates will be discussed. The qicPCR assay results for detecting onset of infection for 53 healthy human volunteers inoculated with RSV will be discussed in the context of plaque assay data and quantitative PCR data.
Section snippets
Subjects and experimental procedures
Healthy volunteers were intra-nasally inoculated with a Good Manufacturing Practise (GMP) clinical strain of RSV-A Memphis 37b and quarantined for 12 days. Nasal washes were collected and analysed by qualitative integrated cycler PCR (qicPCR) twice daily (morning and evening) up to study day 4 and then once on study day 5 morning. Seven independent quarantines were set up over a 3 month period.
Study ethical review and informed consent
The study was approved by the National Research Ethics Service London — City and East (Bristol, United
Validation of qualitative integrated cycler PCR (qicPCR) technology for detection of RSV in clinical saline nasal wash
The limit of detection of RSV in saline by qicPCR was assessed by spiking aliquots of saline media used for clinical nasal wash (NW) sample collection with a ½ log10 dilution series of RSV Memphis 37b ranging from 1000 pfu/mL to 0.3 pfu/mL. The RSV Memphis 37b spiked samples were assayed by multiple operators on different days and utilised 4 different 3 M™ integrated cycler machines (n = 65, Table 1). Samples spiked with100 pfu/mL or greater of RSV were 100% detected by qicPCR. In addition, a high
Discussion
The 3 M™ integrated cycler and CE marked Simplexa™ FluA/B&RSV Direct assay kits for the qicPCR assay have been previously used to assay for RSV viral loads in clinical nasopharyngeal swab samples collected from individuals exhibiting influenza-like illness (Selvaraju et al., 2014). The current study shows that the utility of this assay can be extended to clinical saline NW samples from asymptomatic individuals and can be used as a basis for informed dosing clinical protocols designed to optimize
Acknowledgements
We gratefully acknowledge the contributions of clinical nurses and doctors at Retroscreen who expertly supervised the safety and comfort of the human volunteers in Flucamp and the Translational Research staff who managed the clinical sample handling.
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