Respiratory Syncytial Virus Human Experimental Infection Model: Provenance, Production, and Sequence of Low-Passaged Memphis-37 Challenge Virus

<div><p>Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children and is responsible for as many as 199,000 childhood deaths annually worldwide. To support the development of viral therapeutics and vaccines for RSV, a human adult experimental infection model has been established. In this report, we describe the provenance and sequence of RSV Memphis-37, the low-passage clinical isolate used for the model's reproducible, safe, experimental infections of healthy, adult volunteers. The predicted amino acid sequences for major proteins of Memphis-37 are compared to nine other RSV A and B amino acid sequences to examine sites of vaccine, therapeutic, and pathophysiologic interest. Human T- cell epitope sequences previously defined by <i>in vitro</i> studies were observed to be closely matched between Memphis-37 and the laboratory strain RSV A2. Memphis-37 sequences provide baseline data with which to assess: (i) virus heterogeneity that may be evident following virus infection/transmission, (ii) the efficacy of candidate RSV vaccines and therapeutics in the experimental infection model, and (iii) the potential emergence of escape mutants as a consequence of experimental drug treatments. Memphis-37 is a valuable tool for pre-clinical research, and to expedite the clinical development of vaccines, therapeutic immunomodulatory agents, and other antiviral drug strategies for the protection of vulnerable populations against RSV disease.</p></div

Predicted amino acid sequence for RSV Memphis-37 F protein and alignments.

<p>Alignments are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone-0113100-g001" target="_blank">Figure 1</a>, but for the F protein. Features of interest are shaded or boxed as indicated below the sequence. The blue line marks an antigenic site A, which is targeted by the monoclonal antibody Palivizumab.</p

Comparison of human CD4 and CD8 T-lymphocyte epitope sequences between respiratory syncytial virus Memphis-37 and A2 strains.

<p>Legend: *Differences between Memphis-37 and RSV A2 are bolded.</p><p>Comparison of human CD4 and CD8 T-lymphocyte epitope sequences between respiratory syncytial virus Memphis-37 and A2 strains.</p

Predicted amino acid sequence for RSV Memphis-37 G protein and alignments.

<p>Predicted amino acid sequence is shown for the RSV Memphis-37 G protein. Sequence was aligned with five subtype A viruses (including two laboratory strains) and four subtype B viruses (including two laboratory strains). Virus nomenclature and GenBank Accession numbers used in the alignment are: RSVA Nashville (JX069801.1), RSVA Denver (GU591769.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-Kumaria1" target="_blank">[42]</a>), RSVA Milwaukee (JF920069.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-RebuffoScheer1" target="_blank">[43]</a>), RSVA VR-26 Long (AY911262.1, Laboratory strain), RSVA A2 (M74568.1, Laboratory strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-Stec1" target="_blank">[15]</a>), RSVB Dallas (JQ582843.1), RSVB Milwaukee (JN032117.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-RebuffoScheer1" target="_blank">[43]</a>), RSVB 9320 (AY353550.1, B9320 Laboratory strain), RSV strain B1 (AF013254.1, B1 Laboratory strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-Karron1" target="_blank">[44]</a>). N-terminal variable region: a.a. 67–147, central conserved region: a.a. 148–198, C-terminal hypervariable region: a.a. 199–298, CX3C motif: a.a. 182–186. <u>RSV Memphis-37 sequencing methods</u>: For sequencing purposes, Memphis-37 was taken after five passages and was amplified once more in a T25 flask of HEp-2 cells. Briefly, virus was added to cells in DMEM/0.1% BSA for 1.5 hours. Medium was removed and replaced with EMEM/5% FCS for four days. A lysate was prepared and viral RNA was extracted using a Qiagen viral RNA mini kit. PCR reactions were performed using the TaKaRa One Step RNA PCR Kit (AMV) using 5 µl (145 ng/µl) of viral RNA extracted with Qiagen QIAmp Viral RNA Mini Kit. Forward and reverse oligonucleotides were prepared at concentrations of 100 µM and 1 µl of each pair was used in each reaction. Incubations were at 50°C for 30 min. and 94°C for 2 min. Then 40 cycles were run at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1.5 min. For each sequencing reaction, 40 ng PCR product were mixed with 3.2 pmoles oligonucleotide primer in a final volume of 12 µl and submitted to the Hartwell Center at St. Jude for Sanger sequencing. Sequences were edited and a contig was created in Vector NTI SeqMan. The consensus contig for Memphis-37 was imported into CLC Workbench and predicted amino acid sequences were aligned with other RSV sequences.</p

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

Rationale: Respiratory syncytial virus (RSV) is the leading cause ofchildhood lower respiratory infection, yet viable therapies arelacking. Two major challenges have stalled antiviral development:ethical difficulties in performing pediatric proof-of-concept studiesand the prevailing concept that the disease is immune-mediatedrather than being driven by viral load.Objectives: The development of ahumanexperimental wild-type RSVinfection model to address these challenges.Methods: Healthy volunteers (n 5 35), in five cohorts, receivedincreasing quantities (3.0–5.4 log plaque-forming units/person) ofwild-type RSV-A intranasally.Measurements andMain Results: Overall, 77%of volunteers consistentlyshed virus. Infection rate, viral loads, disease severity, and safety weresimilar between cohorts and were unrelated to quantity of RSV received.Symptomsbegan near the time of initial viral detection, peakedin 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 proinflammatorycytokine concentrations (IL-6 and IL-8). Increased viral load correlatedconsistently with increases inmultiple different diseasemeasurements(symptoms, physical examination, and amount of nasal mucus).Conclusions:Viralloadappears todrive diseasemanifestations inhumanswith RSV infection. The observed parallel viral and disease kineticssupport a potential clinical benefit of RSV antivirals. This reproduciblemodel facilitates the development of future RSV therapeutics

Viral load drives disease in humans experimentally infected with respiratory syncytial virus

Rationale: Respiratory syncytial virus (RSV) is the leading cause ofchildhood lower respiratory infection, yet viable therapies arelacking. Two major challenges have stalled antiviral development:ethical difficulties in performing pediatric proof-of-concept studiesand the prevailing concept that the disease is immune-mediatedrather than being driven by viral load.Objectives: The development of ahumanexperimental wild-type RSVinfection model to address these challenges.Methods: Healthy volunteers (n 5 35), in five cohorts, receivedincreasing quantities (3.0–5.4 log plaque-forming units/person) ofwild-type RSV-A intranasally.Measurements andMain Results: Overall, 77%of volunteers consistentlyshed virus. Infection rate, viral loads, disease severity, and safety weresimilar between cohorts and were unrelated to quantity of RSV received.Symptomsbegan near the time of initial viral detection, peakedin 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 proinflammatorycytokine concentrations (IL-6 and IL-8). Increased viral load correlatedconsistently with increases inmultiple different diseasemeasurements(symptoms, physical examination, and amount of nasal mucus).Conclusions:Viralloadappears todrive diseasemanifestations inhumanswith RSV infection. The observed parallel viral and disease kineticssupport a potential clinical benefit of RSV antivirals. This reproduciblemodel facilitates the development of future RSV therapeutics