Robert Lambkin-Williams

A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively (P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients (P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.

Gene Expression Signatures Diagnose Influenza and Other Symptomatic Respiratory Viral Infections in Humans

Acute respiratory infections (ARIs) are a common reason for seeking medical attention, and the threat of pandemic influenza will likely add to these numbers. Using human viral challenge studies with live rhinovirus, respiratory syncytial virus, and influenza A, we developed peripheral blood gene expression signatures that distinguish individuals with symptomatic ARIs from uninfected individuals with >95% accuracy. We validated this acute respiratory viral signature-encompassing genes with a known role in host defense against viral infections-across each viral challenge. We also validated the signature in an independently acquired data set for influenza A and classified infected individuals from healthy controls with 100% accuracy. In the same data set, we could also distinguish viral from bacterial ARIs (93% accuracy). These results demonstrate that ARIs induce changes in human peripheral blood gene expression that can be used to diagnose a viral etiology of respiratory infection and triage symptomatic individuals.

Viral Load Drives Disease in Humans Experimentally Infected with Respiratory Syncytial Virus

RATIONALEnRespiratory syncytial virus (RSV) is the leading cause of childhood lower respiratory infection, yet viable therapies are lacking. Two major challenges have stalled antiviral development: ethical difficulties in performing pediatric proof-of-concept studies and the prevailing concept that the disease is immune-mediated rather than being driven by viral load.nnnOBJECTIVESnThe development of a human experimental wild-type RSV infection model to address these challenges.nnnMETHODSnHealthy volunteers (n = 35), in five cohorts, received increasing quantities (3.0-5.4 log plaque-forming units/person) of wild-type RSV-A intranasally.nnnMEASUREMENTS AND MAIN RESULTSnOverall, 77% of volunteers consistently shed virus. Infection rate, viral loads, disease severity, and safety were similar between cohorts and were unrelated to quantity of RSV received. Symptoms began near the time of initial viral detection, peaked in severity near when viral load peaked, and subsided as viral loads (measured by real-time polymerase chain reaction) slowly declined. Viral loads correlated significantly with intranasal proinflammatory cytokine concentrations (IL-6 and IL-8). Increased viral load correlated consistently with increases in multiple different disease measurements (symptoms, physical examination, and amount of nasal mucus).nnnCONCLUSIONSnViral load appears to drive disease manifestations in humans with RSV infection. The observed parallel viral and disease kinetics support a potential clinical benefit of RSV antivirals. This reproducible model facilitates the development of future RSV therapeutics.

Oral GS-5806 Activity in a Respiratory Syncytial Virus Challenge Study

BACKGROUNDnRespiratory syncytial virus (RSV) is a common cause of infant hospitalizations and is increasingly recognized as a cause of considerable morbidity and mortality. No accepted antiviral treatment exists.nnnMETHODSnWe conducted a double-blind, placebo-controlled study of GS-5806, an oral RSV-entry inhibitor, in healthy adults who received a clinical challenge strain of RSV intranasally. Participants were monitored for 12 days. At the time of a positive test for RSV infection or 5 days after inoculation, whichever occurred first, participants were randomly assigned to receive GS-5806 or placebo in one of seven sequential cohorts. Cohorts 1 to 4 received a first dose of 50 mg of GS-5806 and then 25 mg daily for the next 4 days, cohort 5 received a first dose of 50 mg and then 25 mg daily for the next 2 days, cohort 6 received one 100-mg dose, and cohort 7 received a first dose of 10 mg and then 5 mg daily for the next 4 days. Dose selection for cohorts 5, 6, and 7 occurred after an interim analysis of data for cohorts 1 to 4. The primary end point was the area under the curve (AUC) for the viral load, which was assessed after administration of the first dose through the 12th day after inoculation. Secondary end points were mucus weight and symptom scores.nnnRESULTSnAmong the 54 participants in cohorts 1 to 4 who were infected with RSV, active treatment was associated with a lower viral load (adjusted mean, 250.7 vs. 757.7 log10 plaque-forming-unit equivalents [PFUe] × hours per milliliter; P<0.001), lower total mucus weight (mean, 6.9 g vs. 15.1 g; P=0.03), and a lower AUC for the change from baseline in symptom scores (adjusted mean, -20.2 vs. 204.9 × hours; P=0.005). The results were similar in cohorts 5, 6, and 7. Adverse events, including low neutrophil counts and increased levels of alanine aminotransferase, were more common among participants receiving GS-5806.nnnCONCLUSIONSnTreatment with GS-5806 reduced the viral load and the severity of clinical disease in a challenge study of healthy adults. (Funded by Gilead Sciences; ClinicalTrials.gov number, NCT01756482.).

A host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza H1N1 or H3N2.

There is great potential for host-based gene expression analysis to impact the early diagnosis of infectious diseases. In particular, the influenza pandemic of 2009 highlighted the challenges and limitations of traditional pathogen-based testing for suspected upper respiratory viral infection. We inoculated human volunteers with either influenza A (A/Brisbane/59/2007 (H1N1) or A/Wisconsin/67/2005 (H3N2)), and assayed the peripheral blood transcriptome every 8 hours for 7 days. Of 41 inoculated volunteers, 18 (44%) developed symptomatic infection. Using unbiased sparse latent factor regression analysis, we generated a gene signature (or factor) for symptomatic influenza capable of detecting 94% of infected cases. This gene signature is detectable as early as 29 hours post-exposure and achieves maximal accuracy on average 43 hours (pu200a=u200a0.003, H1N1) and 38 hours (p-valueu200a=u200a0.005, H3N2) before peak clinical symptoms. In order to test the relevance of these findings in naturally acquired disease, a composite influenza A signature built from these challenge studies was applied to Emergency Department patients where it discriminates between swine-origin influenza A/H1N1 (2009) infected and non-infected individuals with 92% accuracy. The host genomic response to Influenza infection is robust and may provide the means for detection before typical clinical symptoms are apparent.

Animal models in influenza vaccine testing

The threat of a pandemic outbreak of influenza A H5N1 and H2N2 has brought attention to the development of new vaccines. Regulatory authorities require companies to provide data proving the effectiveness of vaccines, which cannot, however, be based on real efficacy data in humans. A weight-of-evidence approach may be used, based on evidence of protection in an appropriate animal model and the satisfaction of the surrogate end points in the clinical situation. In this review, we will discuss various animal species that can be infected with influenza. The main animals used for testing vaccines destined for human use are laboratory mice and ferrets and, to a lesser extent, macaques. We will focus particularly on these species.

Comparing influenza and RSV viral and disease dynamics in experimentally infected adults predicts clinical effectiveness of RSV antivirals.

BACKGROUNDnAntivirals reduce influenza viral replication and illness measures, particularly if initiated early, within 48 h of symptom onset. Whether experimental antivirals that reduce respiratory syncytial virus (RSV) load would also reduce disease is unknown. This study compares viral and disease dynamics in humans experimentally infected with influenza or RSV.nnnMETHODSnClinical strains of RSV-A and influenza A were inoculated intranasally into 20 and 17 healthy volunteers, respectively, on day 0. Symptom scores and nasal washes were performed twice daily, and daily mucus weights were collected. Viral loads in nasal washes were quantified by culture (plaque assay in HEp-2 cells for RSV and by end point dilution in Madin-Darby canine kidney cells for influenza).nnnRESULTSnAfter influenza inoculation, influenza viral load and illness markers increased simultaneously until day 2. Within individual subjects, peak influenza load occurred 0.4 days (95% CI -0.4, 1.3) before peak symptoms. Influenza viral load and disease declined thereafter. After RSV inoculation, a longer incubation period occurred prior to viral detection and symptom onset. RSV load and disease increased together until day 5. Within individual subjects, peak RSV loads occurred 0.2 days (95% CI -0.7, 1.05) before peak symptoms, after which both illness measures and viral load declined together.nnnCONCLUSIONSnViral and disease dynamics in experimental human infections suggest that reducing RSV load, if timed similarly to clinically-effective influenza antivirals, might be expected to have a similar or greater window of opportunity for reducing clinical RSV disease.

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification

ABSTRACT Knowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation. IMPORTANCE Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects viral populations differ genetically from the viral stock, with subjects viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.

Longitudinal analysis of leukocyte differentials in peripheral blood of patients with acute respiratory viral infections

BACKGROUNDnLeukocyte counts and differentials are commonly acquired in patients with suspected respiratory viral infections and may contribute diagnostic information. However, most published work is limited to a single timepoint at initial presentation to a medical provider, which may correspond to widely varying points in the course of disease.nnnOBJECTIVESnTo examine the temporal development and time-dependent utility of routine leukocyte differentials in the diagnosis of respiratory viral infections.nnnSTUDY DESIGNnWe analyzed data from recent experimental human challenges with influenza A/H3N2, human rhinovirus (HRV), and respiratory syncytial virus (RSV). Routine clinical lab cell counts and differentials were measured daily from the time period immediately prior to inoculation through the eventual resolution of symptomatic disease.nnnRESULTSnApproximately 50% of challenged individuals developed symptoms and viral shedding consistent with clinical disease. Subpopulations of WBC showed marked differences between symptomatic and asymptomatic individuals over time, but these changes were much more profound and consistent in influenza infection. Influenza-infected subjects develop both relative lymphopenia and relative monocytosis, both of which closely mirror symptom development in time. A lymphocyte:monocyte ratio of <2 correctly classifies 100% of influenza (but not RSV or HRV) infected subjects at the time of maximal symptoms.nnnCONCLUSIONSnLeukocyte differentials may suggest a viral etiology in patients with upper respiratory infection, but are not sufficient to allow differentiation between common viruses. Timing of data acquisition relative to the disease course is a key component in determining the utility of these tests.

Low pH gel intranasal sprays inactivate influenza viruses in vitro and protect ferrets against influenza infection

BackgroundDeveloping strategies for controlling the severity of pandemic influenza is a global public health priority. In the event of a pandemic there may be a place for inexpensive, readily available, effective adjunctive therapies to support containment strategies such as prescription antivirals, vaccines, quarantine and restrictions on travel. Inactivation of virus in the intranasal environment is one possible approach. The work described here investigated the sensitivity of influenza viruses to low pH, and the activity of low pH nasal sprays on the course of an influenza infection in the ferret model.MethodsInactivation of influenza A and avian reassortment influenza was determined using in vitro solutions tests. Low pH nasal sprays were tested using the ferret model with an influenza A Sydney/5/97 challenge. Clinical measures were shed virus, weight loss and body temperature.ResultsThe virus inactivation studies showed that influenza viruses are rapidly inactivated by contact with acid buffered solutions at pH 3.5. The titre of influenza A Sydney/5/97 [H3N2] was reduced by at least 3 log cycles with one minute contact with buffers based on simple acid mixtures such as L-pyroglutamic acid, succinic acid, citric acid and ascorbic acid. A pH 3.5 nasal gel composition containing pyroglutamic acid, succinic acid and zinc acetate reduced titres of influenza A Hong Kong/8/68 [H3N2] by 6 log cycles, and avian reassortment influenza A/Washington/897/80 X A Mallard/New York/6750/78 [H3N2] by 5 log cycles, with 1 min contact.Two ferret challenge studies, with influenza A Sydney/5/97, demonstrated a reduction in the severity of the disease with early application of low pH nasal sprays versus a saline control. In the first study there was decreased weight loss in the treatment groups. In the second study there were reductions in virus shedding and weight loss, most notably when a gelling agent was added to the low pH formulation.ConclusionThese findings indicate the potential of a low pH nasal spray as an adjunct to current influenza therapies, and warrant further investigation in humans.