Protein kinase D1 (PKD1), a ubiquitously expressed serine/threonine kinase, regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, vesicle trafficking, Ag receptor signaling, and pattern recognition receptor signaling. We found previously that exposure to hypersensitivity pneumonitis (HP) inciting Ag Saccharopolyspora rectivirgula leads to the activation of PKD1 in a MyD88-dependent manner in various types of murine cells in vitro and in the mouse lung in vivo. However, it is currently unknown whether PKD1 plays a role in the S. rectivirgula–induced HP. In this study, we investigated contributions of PKD1 on the S. rectivirgula–induced HP using conditional PKD1-insufficient mice. Compared to control PKD1-sufficient mice, PKD1-insufficient mice showed substantially suppressed activation of MAPKs and NF-κB, expression of cytokines and chemokines, and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula. The significantly reduced levels of alveolitis, MHC class II surface expression on neutrophils and macrophages, and IL-17A and CXCL9 expression in lung tissue were observed in the PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk. PKD1-insuficient mice exposed to S. rectivirgula for 5 wk also showed reduced granuloma formation. Our results demonstrate that PKD1 plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposure to S. rectivirgula and substantially contribute to the development of HP caused by repeated exposure to S. rectivirgula. Our findings suggest that PKD1 can be an attractive new molecular target for therapy of S. rectivirgula–induced HP.

Effective treatments for respiratory syncytial virus (RSV) infection are lacking. Here, we report a human proof-of-concept study for RV521, a small-molecule antiviral inhibitor of the RSV-F protein. In this randomized, double-blind, placebo-controlled trial, healthy adults were challenged with RSV-A Memphis-37b. ABSTRACT Effective treatments for respiratory syncytial virus (RSV) infection are lacking. Here, we report a human proof-of-concept study for RV521, a small-molecule antiviral inhibitor of the RSV-F protein. In this randomized, double-blind, placebo-controlled trial, healthy adults were challenged with RSV-A Memphis-37b. After infection was confirmed (or 5 days after challenge virus inoculation), subjects received RV521 (350 mg or 200 mg) or placebo orally every 12 h for 5 days. The primary endpoint was area under the curve (AUC) for viral load, as assessed by reverse transcriptase quantitative PCR (RT-qPCR) of nasal wash samples. The primary efficacy analysis set included subjects successfully infected with RSV who received ≥1 dose of study drug. A total of 66 subjects were enrolled (n = 22 per group); 53 were included in the primary analysis set (RV521 350 mg: n = 16; 200 mg: n = 18; placebo: n = 19). The mean AUC of RT-qPCR-assessed RSV viral load (log10 PFU equivalents [PFUe]/ml · h) was significantly lower with RV521 350 mg (185.26; standard error [SE], 31.17; P = 0.002) and 200 mg (224.35; SE, 37.60; P = 0.007) versus placebo (501.39; SE, 86.57). Disease severity improved with RV521 350 mg and 200 mg versus placebo (P = 0.002 and P = 0.009, respectively, for AUC total symptom score [score × hours]). Daily nasal mucus weight was significantly reduced (P = 0.010 and P = 0.038 for RV521 350 mg and 200 mg, respectively, versus placebo). All treatment-emergent adverse events were grade 1 or 2. No subjects discontinued due to adverse events. There was no evidence of clinically significant viral resistance, and only three variants were detected. RV521 effectively reduced RSV viral load and disease severity in humans and was well tolerated. (This study has been registered at ClinicalTrials.gov under registration no. NCT03258502.)

The purpose of this study was to develop a more accurate method for analyzing fashion image digital colors in uncertain observation conditions. The standard color values used in this experiment were obtained from 10 pieces of actual clothing provided by their designers. The comparison color values were created from fashion images using human visual perception, software color analysis, and a combination of human visual perception and software color analysis. The other 10 color values were determined using the method developed in this study. The data were processed using Microsoft Excel 2013 and the statistical program R 3.4.2. Descriptive analyses, 1-way anova tests, and a multiple comparisons post-test were conducted using false discovery rates. The results showed that a combination of human and software analysis produced more accurate results than either alone. Delta E-ab* 3-6 was found to be the minimum color difference tolerance between actual clothing and fashion image.

Many aspects of the respiratory syncytial virus (RSV) are still poorly understood. Yet these knowledge gaps have had and could continue to have adverse, unintended consequences for the efficacy and safety of antivirals and vaccines developed against RSV. Mathematical modelling was used to test and evaluate hypotheses about the rate of loss of RSV infectivity and the mechanisms and kinetics of RSV infection spread in SIAT cells in vitro. While the rate of loss of RSV integrity, as measured via qRT-PCR, is well-described by an exponential decay, the latter mechanism failed to describe the rate at which RSV A Long loses infectivity over time in vitro based on the data presented herein. This is unusual given that other viruses (HIV, HCV, influenza) have been shown to lose their infectivity exponentially in vitro, and indeed an exponential rate of loss of infectivity is always assumed in mathematical modelling and experimental analyses. The infectivity profile of RSV in HEp-2 and SIAT cells remained consistent over the course of an RSV infection, over time and a large range of infectivity. However, SIAT cells were found to be ∼ 100× less sensitive to RSV infection than HEp-2 cells. In particular, we found that RSV spreads inefficiently in SIAT cells, in a manner we show is consistent with the establishment of infection resistance in uninfected cells. SIAT cells are a good in vitro model in which to study RSV in vivo dissemination, yielding similar infection timescales. However, the higher sensitivity of HEp-2 cells to RSV together with its RSV infectivity profile being similar to that of SIAT cells, makes HEp-2 cells more suitable for quantifying RSV infectivity over the course of in vitro RSV infections in SIAT cells. Our findings highlight the importance and urgency of resolving the mechanisms at play in the dissemination of RSV infections in vitro, and the processes by which this infectivity is lost.

BackgroundRespiratory syncytial virus (RSV) disease has no effective treatment. JNJ-53718678 is a fusion inhibitor with selective activity against RSV.MethodsAfter confirmation of RSV infection or 5 days after inoculation with RSV, participants (n = 69) were randomized to JNJ-53718678 75 mg (n = 15), 200 mg (n = 17), 500 mg (n = 18), or placebo (n = 17) orally once daily for 7 days. Antiviral effects were evaluated by assessing RSV RNA viral load (VL) area under the curve (AUC) from baseline (before the first dose) until discharge, time-to-peak VL, duration of viral shedding, clinical symptoms, and quantity of nasal secretions.ResultsMean VL AUC was lower for individuals treated with different doses of JNJ-53718678 versus placebo (203.8-253.8 vs 432.8 log10 PFUe.hour/mL). Also, mean peak VL, time to peak VL, duration of viral shedding, mean overall symptom score, and nasal secretion weight were lower in each JNJ-53718678-treated group versus placebo. No clear exposure-response relationship was observed. Three participants discontinued due to treatment-emergent adverse events of grade 2 and 1 electrocardiogram change (JNJ-53718678 75 mg and 200 mg, respectively) and grade 2 urticaria (placebo).ConclusionsJNJ-53718678 at all 3 doses substantially reduced VL and clinical disease severity, thus establishing clinical proof of concept and the compound's potential as a novel RSV treatment.Clinical trials registrationClinicalTrials.gov: NCT02387606; EudraCT number: 2014-005041-41.

BackgroundRespiratory syncytial virus (RSV) viral load and disease severity associate, and the timing of viral load and disease run in parallel. An antiviral must be broadly effective against the natural spectrum of RSV genotypes and must attain concentrations capable of inhibiting viral replication within the human respiratory tract.ObjectivesWe evaluated a novel RSV fusion inhibitor, MDT‐637, and compared it with ribavirin for therapeutic effect in vitro to identify relative therapeutic doses achievable in humans.MethodMDT‐637 and ribavirin were co‐incubated with RSV in HEp‐2 cells. Quantitative PCR assessed viral concentrations; 50% inhibitory concentrations (IC50) were compared to achievable human MDT‐637 and ribavirin peak and trough concentrations.Results and conclusionsThe IC50 for MDT‐637 and ribavirin (against RSV‐A Long) was 1.42 and 16 973 ng/mL, respectively. The ratio of achievable peak respiratory secretion concentration to IC50 was 6041‐fold for MDT‐637 and 25‐fold for aerosolized ribavirin. The ratio of trough concentration to IC50 was 1481‐fold for MDT‐637 and 3.29‐fold for aerosolized ribavirin. Maximal peak and trough levels of oral or intravenous ribavirin were significantly lower than their IC50s. We also measured MDT‐637 IC50s in 3 lab strains and 4 clinical strains. The IC50s ranged from 0.36 to 3.4 ng/mL. Achievable human MDT‐637 concentrations in respiratory secretions exceed the IC50s by factors from hundreds to thousands of times greater than does ribavirin. Furthermore, MDT‐637 has broad in vitro antiviral activity on clinical strains of different RSV genotypes and clades. Together, these data imply that MDT‐637 may produce a superior clinical effect compared to ribavirin on natural RSV infections.

Saccharopolyspora rectivirgula (SR) causes farmers’ lung disease, a form of hypersensitivity pneumonitis (HP). The initial proinflammatory responses to SR is dependent on Toll-like receptors (TLR) 2 and 9, and their signaling adaptor MyD88. We found previously that protein kinase D1 (PKD1), a serine/threonine kinase, can be activated through a MyD88-dependent TLR signaling pathway and plays a pivotal role in TLR/MyD88-dependent production of proinflammatory mediators. SR activates PKD1 via a MyD88-dependent manner in murine cells and SR-induced alveolitis is substantially suppressed by pretreatment with a PKC/PKD inhibitor Gö6976. However, it is currently unknown whether PKD1 plays a regulatory role in SR-mediated proinflammatory responses and alveolitis in vivo. In the present study, to further understand contribution of PKD1 in the SR-induced pulmonary inflammation, tamoxifen-inducible PKD1-deficient mice were generated. Wild-type mice and PKD1-deficient mice were challenged intranasally with SR. We found that the levels of proinflammatory mediators (including TNFα, IL-6, IL-12, and IFNγ), alveolitis, and neutrophil influx in the bronchoalveolar lavage fluid and interstitial lung tissue after single intranasal expose to SR were significantly lower in PKD1-deficient mice compared to those in wild-type mice. Our results demonstrate that PKD1 is essential for SR-mediated proinflammatory responses and neutrophil influx in the lung. Our findings also suggest that PKD1 might be one of the critical factors that contributes to the development of HP, and could be a therapeutic target for certain forms of HP, such as farmers’ lung disease.

Group B streptococcus (GBS) causes life-threatening diseases, like pneumonia, septicemia and meningitis, in neonates. It has been reported that the pathogen-associated molecular patterns in GBS are recognized by pattern-recognition receptors, including Toll-like receptors (TLR), in the host innate immune cells. This recognition results in the cascades of inflammatory reactions. However, the molecular basis of GBS-induced proinflammatory responses are not yet completely elucidated. Protein kinase D1 (PKD1) has been identified as one of key signaling intermediaries that plays an indispensable role in proinflammatory responses mediated by TLRs. However, it is currently unknown whether PKD1 is activated, and contributes to proinflammatory responses induced by GBS. In the present study, the role of PKD1 in the proinflammatory responses to GBS is investigated. We found that both live and antibiotic-killed GBS activate PKD1 in human and murine cells. This PKD1 activation is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IRAK1, but not TRAF6. Inhibition of PKD using a pharmacological inhibitor revealed that PKD1 is indispensable for GBS-mediated expression of various cytokines/chemokines in vitro and in vivo. Furthermore, systemic administration of PKD inhibitor protects D-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. Our findings imply that PKD1 is one of the critical factors that play a regulatory role in GBS-induced proinflammatory reactions, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates might be an effective way to control GBS diseases.

As RNA virus mutation occurs during replication within host cells, we hypothesized that viral evolution during acute infections in healthy hosts reflects host immune pressure. We therefore investigated the within-host diversification of human respiratory syncytial virus (RSV), a highly prevalent cause of acute respiratory infections. We evaluated healthy adults experimentally infected with an identical inoculum and infants hospitalized with naturally acquired infections. In aggregate, viral diversification in adults peaked at day 3, with overrepresentation of diversity in the matrix protein 2 (M2) and non-structural protein 2 (NS2) genes. In one subject, delayed viral clearance was accompanied by a late peak of diversity at day 10 in known and predicted B and T cell epitopes. In contrast, infant infections showed much less viral diversity. Our findings suggest multiple overlapping mechanisms for early control of acute viral infections, which may differ between age groups and host immune responses.

250/250 words) 23 Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory 24 tract infection in infants and young children, attempts to develop an effective therapy have so 25 far proved unsuccessful. Herein we report the preclinical profiles of PC786, a potent non26 nucleoside RSV L-protein polymerase inhibitor, designed for inhalation treatment of RSV 27 infection. PC786 demonstrated a potent and selective anti-viral activity against laboratory 28 adapted or clinical isolates of RSVA (IC50: <0.09 0.71 nM) and RSVB (IC50: 1.3 50.6 nM), 29 which were determined by inhibition of cytopathic effects in HEp-2 cells without causing 30 detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR 31 analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) 32 and were retained in the face of established RSV replication in a time-of-addition study. 33 Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial 34 cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to 35 undetectable levels in lung homogenates from RSV infected mice and cotton rats. Treatment 36 with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in 37 vitro. In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) 38 activity in a cell-free enzyme assay and mini-genome assay in HEp-2 cells (IC50: 2.1 and 0.5 39 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of 40 RdRp activity, making topical treatment with this compound a novel potential therapy for the 41 treatment of human RSV infections. 42 on A uust 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom

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 [IC 50 ], <0.09 to 0.71 nM) and RSV-B (IC 50 , 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 (IC 50 , 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.

Group B streptococci (GBS) are one of the leading causes of life-threatening illness in neonates. Proinflammatory responses to GBS mediated through host innate immune receptors play a critical role in the disease manifestation. However, the mechanisms involved in proinflammatory responses against GBS, as well as the contribution of signaling modulators involved in host immune defense, have not been fully elucidated. In the present study, we investigated the role of protein kinase D (PKD) 1 in the proinflammatory responses to GBS. We found that both live and antibiotic-killed GBS induce activation of PKD1 through a pathway that is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IL-1R-associated kinase 1, but independent of TNFR-associated factor 6. Our studies using pharmacological PKD inhibitors and PKD1-knockdown macrophages revealed that PKD1 is indispensable for GBS-mediated activation of MAPKs and NF-kB and subsequent expression of proinflammatory mediators. Furthermore, systemic administration of a PKD inhibitor protects D-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. These findings imply that PKD1 plays a critical regulatory role in GBS-induced proinflammatory reactions and sepsis, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates could be an effective way to control GBS diseases.

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

BACKGROUND: Viral culture plaque morphology in human cell lines are markers for growth capability and cytopathic effect, and have been used to assess viral fitness and select preattenuation candidates for live viral vaccines. We classified respiratory syncytial virus (RSV) plaque morphology and analyzed the relationship between plaque morphology as compared to subgroup, viral load and clinical severity of infection in infants and children.METHODS: We obtained respiratory secretions from 149 RSV-infected children. Plaque morphology and viral load was assessed within the first culture passage in HEp-2 cells. Viral load was measured by polymerase chain reaction (PCR), as was RSV subgroup. Disease severity was determined by hospitalization, length of stay, intensive care requirement, and respiratory failure.RESULTS: Plaque morphology varied between individual subjects; however, similar results were observed among viruses collected from upper and lower respiratory tracts of the same subject. Significant differences in plaque morphology were observed between RSV subgroups. No correlations were found among plaque morphology and viral load. Plaque morphology did not correlate with disease severity.CONCLUSION: Plaque morphology measures parameters that are viral-specific and independent of the human host. Morphologies vary between patients and are related to, RSV subgroup. In HEp-2 cells, RSV plaque morphology appears unrelated to disease severity in RSV-infected children.

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 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.

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.

While the factors that initiate and perpetuate inflammation in rheumatoid arthritis (RA) are not well understood, it is thought that RA may be initiated in part by signaling through Toll-like receptors (TLRs). Disruption of the TLR signaling pathway may provide an opportunity to halt the RA process in the earliest stages of development. We previously found that protein kinase D1 (PKD1) is essential for proinflammatory responses mediated by MyD88-dependent TLRs. Here, we investigated whether PKD1 is involved in exaggerated proinflammatory responses seen in RA patients, and in the development of experimental arthritis. PKD1 was constitutively hyperactivated in synoviocytes of RA patients, and was rapidly activated in synoviocytes of normal donors and RA patients in response to TLR/IL-1R ligands. Silencing PKD1 expression in RA synoviocytes resulted in substantial inhibition of both spontaneous and TLR/IL-1R-mediated expression of cytokines/chemokines. Suppression of PKD1 expression also resulted in inhibition of TLR/IL-1R-mediated cytokine production in IL-1R antagonist-deficient (IL-1rn-/-) macrophages. In addition, daily treatment with a PKD inhibitor substantially reduced the incidence and severity of spontaneously occurring inflammatory arthritis in IL-1rn-/- mice, and collagen-induced arthritis in humanized HLA-DR1 mice. This implies that PKD1 might be one of the key regulatory factors that modulate proinflammatory responses in RA, and can be a therapeutic target for RA.

Abstract Respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections and hospitalization in infants, but no vaccine exists, primarily because the immune response is poorly understood and immune correlates of protection have not been adequately defined. Antiviral lymphocytes, along with secreted immunoglobulins (Ig) are likely the most important components of effective anti-RSV immunity RSV-specific assays to quantify the effective cell-mediated immune response to naturally-occurring RSV infections are imperative. We therefore quantified IFN-γ secreting human peripheral blood mononuclear cells (PBMCs) with and without stimulation by whole RSV from different clades, and with highly purified RSV-peptide pools. PBMCs from donors demonstrated between-donor variable levels of immune response to the RSV peptide pools and the whole virus preparations. Robust IFN-γ T-cell responses were observed at 72 hours in all PBMCs, thus allowing a single time point assay evaluation. Higher responses were seen in the CD4+ population of PBMCs compared to the CD8+ population. Responses of CD8+ PBMCs to whole virus preparations and to mock infection control preparations were similar and responses to RSV peptide pools were not statistically significant from the negative peptide control. IFN-γ based ELISPOTS and FACS assays are promising tools to quantify RSV-specific T-cell-mediated immune responses in human blood.