John P. DeVincenzo

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

Limited Type I Interferons and Plasmacytoid Dendritic Cells during Neonatal Respiratory Syncytial Virus Infection Permit Immunopathogenesis upon Reinfection

ABSTRACT Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action. IMPORTANCE Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design.

Vaccination strategies against respiratory syncytial virus

Significance The WHO estimates that respiratory syncytial virus (RSV) vaccination will be available in the next 5–10 y. To evaluate the population effectiveness of an RSV vaccination program in the United States, we developed a transmission model that integrates data on daily infectious viral load and behavior changes while symptomatic. Our model simulations demonstrate that vaccinating children younger than 5 y of age will be the most efficient and effective way to prevent RSV infection in both children and older adults, a result that is robust across the US states considered. Accordingly, the population burden of RSV would be most effectively reduced if current vaccine candidates were to focus on children. Respiratory syncytial virus (RSV) is the most common cause of US infant hospitalization. Additionally, RSV is responsible for 10,000 deaths annually among the elderly across the United States, and accounts for nearly as many hospitalizations as influenza. Currently, several RSV vaccine candidates are under development to target different age groups. To evaluate the potential effectiveness of age-specific vaccination strategies in averting RSV incidence, we developed a transmission model that integrates data on daily infectious viral load and changes of behavior associated with RSV symptoms. Calibrating to RSV weekly incidence rates in Texas, California, Colorado, and Pennsylvania, we show that in all states considered, an infected child under 5 y of age is more than twice as likely as a person over 50 y of age to transmit the virus. Geographic variability in the effectiveness of a vaccination program across states arises from interplay between seasonality patterns, population demography, vaccination uptake, and vaccine mechanism of action. Regardless of these variabilities, our analysis showed that allocating vaccine to children under 5 y of age would be the most efficient strategy per dose to avert RSV in both children and adults. Furthermore, due to substantial indirect protection, the targeting of children is even predicted to reduce RSV in the elderly more than directly vaccinating the elderly themselves. Our results can help inform ongoing clinical trials and future recommendations on RSV vaccination.

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

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 models 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 in vitro 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.

Sendai virus-based RSV vaccine protects against RSV challenge in an in vivo maternal antibody model.

Respiratory syncytial virus (RSV) is the cause of significant morbidity and mortality among infants, and despite decades of research there remains no licensed vaccine. SeVRSV is a Sendai virus (SeV)-based live intranasal vaccine that expresses the full length RSV fusion (F) gene. SeV is the murine counterpart of human parainfluenza virus type 1. Given that the target population of SeVRSV is young infants, we questioned whether maternal antibodies typical of this age group would inhibit SeVRSV vaccine efficacy. After measuring SeV- and RSV-specific serum neutralizing antibody titers in human infants, we matched these defined titers in cotton rats by the passive transfer of polyclonal or monoclonal antibody products. Animals were then vaccinated with SeVRSV followed by a 3 month rest period to allow passively transferred antibodies to wane. Animals were finally challenged with RSV to measure the de novo vaccine-induced immune responses. Despite the presence of passively-transferred serum neutralizing antibodies at the time of vaccination, SeVRSV induced immune responses that were protective against RSV challenge. The data encourage advancement of SeVRSV as a candidate vaccine for the protection of children from morbidity and mortality caused by RSV.

Respiratory Mucosal Proteome Quantification in Human Influenza Infections.

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 28 and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection.

Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

ABSTRACT Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC50], <0.09 to 0.71 nM) and RSV-B (IC50, 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro. In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC50, 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections.

Multi-center evaluation of the adenovirus R-gene US assay for the detection of adenovirus in respiratory samples

BACKGROUND Adenoviruses (AdV) cause a variety of upper and lower respiratory tract infections, with the potential for severe outcomes, especially in persons with immune suppression or other underlying diseases. The ADENOVIRUS US R-gene (AdV R-gene, Argene/bioMérieux) is a FDA cleared real-time PCR assay that utilizes primers and fluorescent probes that target a conserved region of the hexon gene and an internal control DNA. OBJECTIVES This prospective multi-center study evaluated the clinical performance of AdV R-gene for AdV detection in respiratory specimens from symptomatic patients of all ages. STUDY DESIGN Nucleic acids from nasopharyngeal washes/aspirates (NPW/A; n=393) and NP flocked swabs (NPS, Copan) (n=1183) were extracted using NucliSENS easyMAG (bioMérieux) and AdV R-gene PCR was performed using the SmartCycler (Cepheid). AdV R-gene results were compared to R-Mix culture (Quidel/Diagnostic Hybrids). For a subset of samples (n=946) AdV R-gene and R-Mix results were also compared to A549 cell culture. RESULTS In first intention analysis for NPS the AdV R-gene positive percent agreement (PPA), and negative percent agreement (NPA) were 91.7% and 96.2%, respectively, and for NPW/A were 100% and 94.4%, respectively, compared to R-Mix culture. In second intention analysis, discordant samples only were tested with an AdV real-time PCR assay (Viracor-IBT Labs) and amplicon sequencing. For NPS, the sensitivity, specificity, PPV and NPV for AdV R-gene were 98.9%, 100%, 100%, and 99.9%, respectively and for R-Mix culture were 51.7%, 99.7%, 93.8%, and 96.3%, respectively. For NPW/A, the sensitivity, specificity, PPV and NPV for AdV R-gene were 100%, 99.7%, 97.6%, and 100%, respectively, and for R-Mix culture were 52.5%, 100%, 100%, and 94.9%, respectively. Overall, AdV was detected by AdV R-gene and R-Mix in 7.4% and 4.1% NPS, respectively, and in 10.7% and 5.3% NPW/A, respectively. Children 5yr and younger had the highest rates of AdV infections. In a subset of specimens (n=946) the sensitivity of AdV R-gene, R-Mix, and A549 cell culture were 95.0%, 55.4% and 66.3%. CONCLUSIONS AdV R-gene is sensitive and specific for the detection of AdV in NPW/A and NPS samples. AdV R-gene is simple to use and provides a rapid time to results (within 2.5-3h).

Unrecognized prolonged viral replication in the pathogenesis of human RSV infection

BACKGROUND Respiratory symptoms in RSV persist long after the virus is no longer detected by culture. Current concepts of RSV pathogenesis explain this by RSV inducing a long-lasting pathogenic immune cascade. We alternatively hypothesized that prolonged unrecognized RSV replication may be responsible and studied this possibility directly in a human wild-type RSV experimental infection model. OBJECTIVE The objective of the current report was to define the duration of true human RSV replication by studying it directly in immunocompetent adults experimentally infected with a clinical strain of RSV utilizing this previously established safe and reproducible model. STUDY DESIGN 35 healthy adult volunteers were inoculated with RSV-A (Memphis-37, a low11 passage clinical strain virus, manufactured from a hospitalized bronchiolitic infant) and evaluated over 12 days. Viral load by culture, parallel quantitative PCR (genomic, message) and RSV-specific IgA, were measured twice daily from serially collected nasal washes. RESULTS After inoculation, 77% (27/35) of volunteers became RSV infected. As expected, culture-detectable RSV ceased abruptly by the 5-6 t h 15 infection day. However, infected volunteers demonstrated prolonged RSV presence by both genomic and message PCR. RSV-specific IgA rose within respiratory secretions of infected volunteers during same time frame. CONCLUSIONS RSV replication appears to continue in humans far longer than previously thought. The rise in nasal RSV-specific IgA shortly after infection likely neutralizes culture detectable virus producing misleadingly short durations of infection. Prolonged viral replication helps explain RSVs extended disease manifestations and increases the potential utility of antivirals.

Traumatic Lumbar Puncture is Unlikely to Reduce Modern Molecular Detection of HSV Encephalitis (HSVE)

Polymerase chain reaction (PCR) is the gold-standard for diagnosing HSVE, but may be inhibited by blood contamination. We mimicked traumatic lumbar puncture and measured its effect on the molecular detection of HSV-1 in CSF. Clinically meaningful reductions in sensitivity were not observed. Pre-PCR processing allows sensitive detection of HSVE despite traumatic lumbar puncture.