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1.  Safety and Immunogenicity of High Dose Trivalent Inactivated Influenza Vaccine in Pediatric Patients with Acute Lymphoblastic Leukemia 
Pediatric blood & cancer  2013;61(5):815-820.
Background
Although children with acute lymphoblastic leukemia (ALL) mount immune responses after vaccination with the trivalent influenza vaccine (TIV), these responses are lower compared to controls. Recently, a high dose (HD) TIV was found to increase the level of antibody response in elderly patients compared to the standard dose (SD) TIV. We hypothesized that the HD TIV would be well-tolerated and more immunogenic compared to the SD TIV in pediatric subjects with ALL.
Procedure
This was a randomized, double-blind, phase I safety and immunogenicity trial comparing the HD to the SD TIV in children with ALL. Subjects were randomized 2:1 to receive either the HD (60µg) or the SD (15µg) TIV. Local and systemic reactions were solicited, hemagglutinin inhibition titers to influenza virus antigens were measured, and monitoring labs were collected prior to and/or after each vaccination.
Results
Fifty subjects were enrolled (34 HD, 16 SD). Mean age was 8.5 years; 63% were male, and 80% were in maintenance therapy. There were no significant differences reported in local or systemic symptoms. No severe adverse events were attributed to vaccination. No significant differences between the HD and SD TIV groups were noted for immune responses.
Conclusions
No differences were noted between the HD and SD TIV groups for solicited systemic and local reactions. Since this study was not powered for immunogenicity, a phase II trial is needed to determine the immunogenicity of HD versus SD TIV in the pediatric ALL population.
doi:10.1002/pbc.24863
PMCID: PMC4310469  PMID: 24249544
Children; Influenza; Leukemia; Trivalent Inactivated Influenza Vaccine
2.  Viral suppressors of the RIG-I-mediated interferon response are pre-packaged in influenza virions 
Nature Communications  2014;5:5645.
The type I interferon (IFN) response represents the first line of defence to invading pathogens. Internalized viral ribonucleoproteins (vRNPs) of negative-strand RNA viruses induce an early IFN response by interacting with retinoic acid inducible gene I (RIG-I) and its recruitment to mitochondria. Here we employ three-dimensional stochastic optical reconstruction microscopy (STORM) to visualize incoming influenza A virus (IAV) vRNPs as helical-like structures associated with mitochondria. Unexpectedly, an early IFN induction in response to vRNPs is not detected. A distinct amino-acid motif in the viral polymerases, PB1/PA, suppresses early IFN induction. Mutation of this motif leads to reduced pathogenicity in vivo, whereas restoration increases it. Evolutionary dynamics in these sequences suggest that completion of the motif, combined with viral reassortment can contribute to pandemic risks. In summary, inhibition of the immediate anti-viral response is ‘pre-packaged’ in IAV in the sequences of vRNP-associated polymerase proteins.
It is unclear how incoming influenza viruses counteract the cells’ first line of defence, the interferon (IFN) response. Here Liedmann et al. show that a distinct amino-acid motif in polymerases PB1 and PA, which are packaged in the viral particles, inhibit early IFN induction.
doi:10.1038/ncomms6645
PMCID: PMC4268707  PMID: 25487526
3.  Molecular signatures of virulence in the PB1-F2 proteins of H5N1 influenza viruses 
Virus research  2013;178(1):146-150.
The PB1-F2 protein of influenza A viruses contributes to pathogenesis in animal models. Specific molecular signatures of virulence within PB1-F2 have been mapped for some functions. The 66S polymorphism may modulate interferon activity, and four C-terminal amino acids, 62L, 75R, 79R, and 82L, contribute to cytokine release and inflammatory responses in specific virus backgrounds. All available PB1-F2 sequences from H5N1 subtype influenza A viruses were analyzed. The majority (82.5%) of H5N1 sequences available in the Influenza Research Database code for PB1-F2 proteins with 4 or more of these virulence associated amino acids. Most of these are avian sequences from highly pathogenic strains isolated in Asia or Africa. The 66S polymorphism was uncommon (5.3% of sequences) but was found in association with the other 4 inflammatory amino acids in select highly pathogenic strains in Asia. These analyses suggest that if an H5N1 virus were to emerge as a pandemic strain, the PB1-F2 protein will be a contributor to pathogenesis. Research on the pathogenic effect of these signatures in an H5N1 background should be undertaken. Surveillance efforts should include sequencing of the PB1 gene segment and analysis for these molecular signatures to allow for the potential prioritization of resources during pandemic planning.
doi:10.1016/j.virusres.2013.02.012
PMCID: PMC4006689  PMID: 23499672
influenza virus; PB1-F2; avian influenza; virulence; H5N1
4.  Non-Avian Animal Reservoirs Present a Source of Influenza A PB1-F2 Proteins with Novel Virulence-Enhancing Markers 
PLoS ONE  2014;9(11):e111603.
PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.
doi:10.1371/journal.pone.0111603
PMCID: PMC4219726  PMID: 25368997
5.  The immune profile associated with acute allergic asthma accelerates clearance of influenza virus 
Immunology and cell biology  2014;92(5):449-459.
Asthma was the most common co-morbidity in hospitalized patients during the 2009 influenza pandemic. For unknown reasons, hospitalized asthmatics had less severe outcomes and were less likely to die from pandemic influenza. Our data with primary human bronchial cells indicate that changes intrinsic to epithelial cells in asthma may protect against cytopathology induced by influenza virus. To further study influenza virus pathogenesis in allergic hosts, we aimed to develop and characterize murine models of asthma and influenza co-morbidity to determine structural, physiological, and immunological changes induced by influenza in the context of asthma. Aspergillus fumigatus-sensitized and -challenged C57BL/6 mice were infected with pandemic H1N1 influenza virus, either during peak allergic inflammation or during airway remodeling to gain insight into disease pathogenesis. Mice infected with influenza during peak allergic inflammation did not lose body weight and cleared the virus rapidly. These mice exhibited high eosinophilia, airway epithelial cell integrity, increased mucus, reduced interferon response, and increased insulin-like growth factor-1. In contrast, weight loss and viral replication curves in the mice that were infected during the late airway remodeling phase were equivalent to flu-only controls. These mice had neutrophils in the airways, damaged airway epithelial cells, less mucus production, increased interferons and decreased insulin-like growth factor-1. The state of the allergic airways at the time of influenza virus infection alters host responses against the virus. These murine models of asthma and influenza co-morbidity may improve our understanding of the epidemiology and pathogenesis of viral infections in humans with asthma.
doi:10.1038/icb.2013.113
PMCID: PMC4037497  PMID: 24469764
Acute asthma; Airway epithelium; Chronic asthma; Co-morbidity; Mouse model
6.  A Novel Cytotoxic Sequence Contributes to Influenza A Viral Protein PB1-F2 Pathogenicity and Predisposition to Secondary Bacterial Infection 
Journal of Virology  2014;88(1):503-515.
Enhancement of cell death is a distinguishing feature of H1N1 influenza virus A/Puerto Rico/8/34 protein PB1-F2. Comparing the sequences (amino acids [aa] 61 to 87 using PB1-F2 amino acid numbering) of the PB1-F2-derived C-terminal peptides from influenza A viruses inducing high or low levels of cell death, we identified a unique I68, L69, and V70 motif in A/Puerto Rico/8/34 PB1-F2 responsible for promotion of the peptide's cytotoxicity and permeabilization of the mitochondrial membrane. When administered to mice, a 27-mer PB1-F2-derived C-terminal peptide with this amino acid motif caused significantly greater weight loss and pulmonary inflammation than the peptide without it (due to I68T, L69Q, and V70G mutations). Similar to the wild-type peptide, A/Puerto Rico/8/34 elicited significantly higher levels of macrophages, neutrophils, and cytokines in the bronchoalveolar lavage fluid of mice than its mutant counterpart 7 days after infection. Additionally, infection of mice with A/Puerto Rico/8/34 significantly enhanced the levels of morphologically transformed epithelial and immune mononuclear cells recruited in the airways compared with the mutant virus. In the mouse bacterial superinfection model, both peptide and virus with the I68, L69, and V70 sequence accelerated development of pneumococcal pneumonia, as reflected by increased levels of viral and bacterial lung titers and by greater mortality. Here we provide evidence suggesting that the newly identified cytotoxic sequence I68, L69, and V70 of A/Puerto Rico/8/34 PB1-F2 contributes to the pathogenesis of both primary viral and secondary bacterial infections.
doi:10.1128/JVI.01373-13
PMCID: PMC3911746  PMID: 24173220
7.  Live Attenuated Influenza Vaccine Enhances Colonization of Streptococcus pneumoniae and Staphylococcus aureus in Mice 
mBio  2014;5(1):e01040-13.
ABSTRACT
Community interactions at mucosal surfaces between viruses, like influenza virus, and respiratory bacterial pathogens are important contributors toward pathogenesis of bacterial disease. What has not been considered is the natural extension of these interactions to live attenuated immunizations, and in particular, live attenuated influenza vaccines (LAIVs). Using a mouse-adapted LAIV against influenza A (H3N2) virus carrying the same mutations as the human FluMist vaccine, we find that LAIV vaccination reverses normal bacterial clearance from the nasopharynx and significantly increases bacterial carriage densities of the clinically important bacterial pathogens Streptococcus pneumoniae (serotypes 19F and 7F) and Staphylococcus aureus (strains Newman and Wright) within the upper respiratory tract of mice. Vaccination with LAIV also resulted in 2- to 5-fold increases in mean durations of bacterial carriage. Furthermore, we show that the increases in carriage density and duration were nearly identical in all aspects to changes in bacterial colonizing dynamics following infection with wild-type (WT) influenza virus. Importantly, LAIV, unlike WT influenza viruses, had no effect on severe bacterial disease or mortality within the lower respiratory tract. Our findings are, to the best of our knowledge, the first to demonstrate that vaccination with a live attenuated viral vaccine can directly modulate colonizing dynamics of important and unrelated human bacterial pathogens, and does so in a manner highly analogous to that seen following wild-type virus infection.
IMPORTANCE
Following infection with an influenza virus, infected or recently recovered individuals become transiently susceptible to excess bacterial infections, particularly Streptococcus pneumoniae and Staphylococcus aureus. Indeed, in the absence of preexisting comorbidities, bacterial infections are a leading cause of severe disease during influenza epidemics. While this synergy has been known and is well studied, what has not been explored is the natural extension of these interactions to live attenuated influenza vaccines (LAIVs). Here we show, in mice, that vaccination with LAIV primes the upper respiratory tract for increased bacterial growth and persistence of bacterial carriage, in a manner nearly identical to that seen following wild-type influenza virus infections. Importantly, LAIV, unlike wild-type virus, did not increase severe bacterial disease of the lower respiratory tract. These findings may have consequences for individual bacterial disease processes within the upper respiratory tract, as well as bacterial transmission dynamics within LAIV-vaccinated populations
doi:10.1128/mBio.01040-13
PMCID: PMC3944816  PMID: 24549845
8.  A live-attenuated pneumococcal vaccine elicits CD4+ T-cell dependent class switching and provides serotype independent protection against acute otitis media 
EMBO Molecular Medicine  2013;6(1):141-154.
Acute otitis media (AOM) caused by Streptococcus pneumoniae remains one of the most common infectious diseases worldwide despite widespread vaccination. A major limitation of the currently licensed pneumococcal vaccines is the lack of efficacy against mucosal disease manifestations such as AOM, acute bacterial sinusitis and pneumonia. We sought to generate a novel class of live vaccines that (1) retain all major antigenic virulence proteins yet are fully attenuated and (2) protect against otitis media. A live vaccine candidate based on deletion of the signal recognition pathway component ftsY induced potent, serotype-independent protection against otitis media, sinusitis, pneumonia and invasive pneumococcal disease. Protection was maintained in animals coinfected with influenza virus, but was lost if mice were depleted of CD4+ T cells at the time of vaccination. The live vaccine induced a strong serum IgG2a and IgG2b response that correlated with CD4+ T-cell mediated class switching. Deletion of genes required for microbial adaptation to the host environment is a novel live attenuated vaccine strategy yielding the first experimental vaccine effective against pneumococcal otitis media.
doi:10.1002/emmm.201202150
PMCID: PMC3936495  PMID: 24408968
otitis media; Streptococcus; vaccine; virulence
9.  Risk of catheter-associated infection in young hematology/oncology patients receiving long-term peripheral nerve blocks 
Paediatric anaesthesia  2012;10.1111/j.1460-9592.2012.03880.x.
Background
Continuous peripheral nerve blocks (CPNBs) are increasingly used to control postoperative and chronic pain. At our pediatric oncology institution, the duration of CPNBs is often prolonged. The risk of catheter-associated infection with prolonged CPNBs has not been previously investigated.
Aim
We analyzed the incidence of CPNB-related infection and its relation to catheter duration, catheter site, intensive care stay, and antibiotic coverage.
Methods
All CPNBs placed at our institution between August 1, 2005 and October 31, 2010 were studied. Primary diagnosis and the site, indication, duration, and infectious adverse effects of CPNBs were obtained from our Pain Service QI database. Patients’ age and sex, antibiotic administration, and number of days in intensive care were collected from patients’ medical records.
Results
The use of 179 catheters in 116 patients was evaluated. Mean age at CPNB placement was 15.1 years (median 14.8, range 0.4–26.9). The most frequent indication for CPNB was surgery (89.4%), most commonly orthopedic (78.8%). Mean CPNB duration was 7.2 days (median 5.0, range 1–81 days). Two cases (1.12%) of CPNBs developed signs of infection, both associated with femoral catheters. The infections were mild and necessitated catheter removal at days 10 and 13, respectively.
Conclusion
Nerve block catheter-associated infections are infrequent at our institution despite prolonged CPNB use. Both patients with infection had femoral catheters and prolonged catheter (≥ 10 days) use.
doi:10.1111/j.1460-9592.2012.03880.x
PMCID: PMC3422424  PMID: 22587819
Peripheral nerve block; infection; oncology; pain
10.  No evidence of a link between influenza vaccines and Guillain-Barre syndrome–associated antiganglioside antibodies 
Background
Guillain-Barre syndrome (GBS) is a rare autoimmune disease characterized by acute, progressive peripheral neuropathy and is commonly associated with the presence of antiganglioside antibodies. Previously, influenza vaccination was linked with the increased incidence of GBS; however, whether antiganglioside antibodies are subsequently induced remains unresolved.
Methods
Sera from human subjects vaccinated with seasonal influenza vaccines from the 2007–2008, 2008–2009, or 1976–1977 influenza seasons were screened for the induction of immunity to influenza and the presence of antiganglioside antibodies pre- and post-vaccination. Likewise, sera from mice vaccinated with seasonal influenza vaccines (1988–1989, 2007–2008) or “swine flu” pandemic vaccines (1976, 2009) were assessed in the same manner. Viruses were also screened for cross-reacting ganglioside epitopes.
Results
Antiganglioside antibodies were found to recognize influenza viruses; this reactivity correlated with virus glycosylation. Antibodies to influenza viruses were detected in human and mouse sera, but the prevalence of antiganglioside antibodies were extremely low.
Conclusions
Although the correlation between antiganglioside antibody cross-reactivity and glycosylation of viruses suggests the role of shared carbohydrate epitopes, no correlation was observed between hemagglutinin-inhibition titers and the induction of antiganglioside antibodies after influenza vaccination.
doi:10.1111/j.1750-2659.2011.00294.x
PMCID: PMC3595170  PMID: 21955390
antibodies; gangliosides; Guillain-Barre syndrome; influenza; vaccines
11.  T-cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection 
Science translational medicine  2012;4(128):128ra42.
A diverse T-cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity by counteracting escape mutations, selecting high-avidity T cells, and providing T-cell specificities with comprehensive functional characteristics. However, evidence that TCR diversity is important for the successful control of human infections is limited. A single-cell strategy for the clonotypic analysis of human CD8+ TCRαβ repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8+ T-cells recovered directly ex vivo. We found that CD8+ TCRαβ repertoire diversity, but not the size of the CD8+ T-cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, our findings indicate that CD8+ T-cell diversity may be more important than T-cell abundance in limiting the negative consequences of CMV persistence, demonstrate high prevalence of both TCRα and β public motif usage, and suggest that a highly diverse TCRαβ repertoire may be an important benchmark and target in the success of immunotherapeutic strategies.
doi:10.1126/scitranslmed.3003647
PMCID: PMC3593633  PMID: 22491952
12.  Kinetics of Coinfection with Influenza A Virus and Streptococcus pneumoniae 
PLoS Pathogens  2013;9(3):e1003238.
Secondary bacterial infections are a leading cause of illness and death during epidemic and pandemic influenza. Experimental studies suggest a lethal synergism between influenza and certain bacteria, particularly Streptococcus pneumoniae, but the precise processes involved are unclear. To address the mechanisms and determine the influences of pathogen dose and strain on disease, we infected groups of mice with either the H1N1 subtype influenza A virus A/Puerto Rico/8/34 (PR8) or a version expressing the 1918 PB1-F2 protein (PR8-PB1-F2(1918)), followed seven days later with one of two S. pneumoniae strains, type 2 D39 or type 3 A66.1. We determined that, following bacterial infection, viral titers initially rebound and then decline slowly. Bacterial titers rapidly rise to high levels and remain elevated. We used a kinetic model to explore the coupled interactions and study the dominant controlling mechanisms. We hypothesize that viral titers rebound in the presence of bacteria due to enhanced viral release from infected cells, and that bacterial titers increase due to alveolar macrophage impairment. Dynamics are affected by initial bacterial dose but not by the expression of the influenza 1918 PB1-F2 protein. Our model provides a framework to investigate pathogen interaction during coinfections and to uncover dynamical differences based on inoculum size and strain.
Author Summary
Influenza virus infected individuals often become coinfected with a bacterial pathogen and, consequently, morbidity and mortality are significantly increased. A better understanding of how these pathogens interact with each other and the host is of key importance. Here, we use data from infected mice together with mathematical modeling and quantitative analyses to understand how each pathogen influences the other, and how the 1918 influenza PB1-F2 protein and the bacterial strain and dose contribute to coinfection kinetics. We find that influenza viral titers increase when Streptococcus pneumoniae is present and that the bacteria establish and grow rapidly when influenza is present. Our model and analyses suggest that the influenza infection reduces the bacterial clearance ability of alveolar macrophages and that the subsequent S. pneumoniae infection enhances viral release from infected cells. These results provide new insights into the mechanisms of influenza coinfection and the differences in pathogenesis of influenza and S. pneumoniae strains.
doi:10.1371/journal.ppat.1003238
PMCID: PMC3605146  PMID: 23555251
13.  Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens 
Journal of Virology  2012;86(17):9035-9043.
A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.
doi:10.1128/JVI.00369-12
PMCID: PMC3416121  PMID: 22674997
14.  Healthcare-Associated Infections at a Children's Cancer Hospital, 1983–2008 
Background.
Little is known about the incidence and etiology of healthcare-associated infections in immunosuppressed children.
Methods.
Data collected prospectively between 1983 and 2008 were used to analyze changes in the rate, types of infection, and infecting organisms over time in patients treated at a children's cancer hospital. Neutropenia was evaluated as a risk factor.
Results.
Over the 26-year study period, 1986 healthcare-associated infections were identified during 1653 hospitalizations. The infection rate decreased significantly from 5.6 to 2.0 infections per 100 discharges (P < .01) and from 9.0 to 3.7 infections per 1000 patient-days (P < .01). Bloodstream infections were the most common type of infection (32.7% of all infections). Staphylococci (46.4% of Gram-positive bacteria), Escherichia coli (36.7% of Gram-negative bacteria), and Candida spp. (68.7% of fungi) were the most common pathogens isolated. An absolute neutrophil count (ANC) nadir <100 per mm3 was significantly associated (P < .0001) with an increased rate of infections compared with higher ANC nadirs.
Conclusions.
Despite a steady expansion in hospital capacity and patient encounters over the last 3 decades, rates of healthcare-associated infections decreased significantly at our hospital. These data suggest that sustained decreases in the rate of healthcare-associated infections in immunosuppressed children are possible. An ANC <100 per mm3 is a risk factor for healthcare-associated infections in this population.
doi:10.1093/jpids/pis034
PMCID: PMC3656547  PMID: 23687571
15.  Receptor-binding specificity of the human parainfluenza virus type 1 hemagglutinin–neuraminidase glycoprotein 
Glycobiology  2011;22(2):174-180.
The hemagglutinin–neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewisx motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.
doi:10.1093/glycob/cwr112
PMCID: PMC3255505  PMID: 21846691
binding; glycan array; hemagglutinin–neuraminidase; parainfluenza; receptor
16.  Impaired Wound Healing Predisposes Obese Mice to Severe Influenza Virus Infection 
The Journal of Infectious Diseases  2011;205(2):252-261.
(See the editorial commentary by Beck, on pages 172–3, and the article by Kim et al, on pages 244–51.)
For the first time, obesity appeared as a risk factor for developing severe 2009 pandemic influenza infection. Given the increase in obesity, there is a need to understand the mechanisms underlying poor outcomes in this population. In these studies, we examined the severity of pandemic influenza virus in obese mice and evaluated antiviral effectiveness. We found that genetically and diet-induced obese mice challenged with either 2009 influenza A virus subtype H1N1 or 1968 subtype H3N2 strains were more likely to have increased mortality and lung pathology associated with impaired wound repair and subsequent pulmonary edema. Antiviral treatment with oseltamivir enhanced survival of obese mice. Overall, these studies demonstrate that impaired wound lung repair in the lungs of obese animals may result in severe influenza virus infection. Alternative approaches to prevention and control of influenza may be needed in the setting of obesity.
doi:10.1093/infdis/jir729
PMCID: PMC3244366  PMID: 22147799
17.  The age distribution of mortality due to influenza: pandemic and peri-pandemic 
BMC Medicine  2012;10:162.
Background
Pandemic influenza is said to 'shift mortality' to younger age groups; but also to spare a subpopulation of the elderly population. Does one of these effects dominate? Might this have important ramifications?
Methods
We estimated age-specific excess mortality rates for all-years for which data were available in the 20th century for Australia, Canada, France, Japan, the UK, and the USA for people older than 44 years of age. We modeled variation with age, and standardized estimates to allow direct comparison across age groups and countries. Attack rate data for four pandemics were assembled.
Results
For nearly all seasons, an exponential model characterized mortality data extremely well. For seasons of emergence and a variable number of seasons following, however, a subpopulation above a threshold age invariably enjoyed reduced mortality. 'Immune escape', a stepwise increase in mortality among the oldest elderly, was observed a number of seasons after both the A(H2N2) and A(H3N2) pandemics. The number of seasons from emergence to escape varied by country. For the latter pandemic, mortality rates in four countries increased for younger age groups but only in the season following that of emergence. Adaptation to both emergent viruses was apparent as a progressive decrease in mortality rates, which, with two exceptions, was seen only in younger age groups. Pandemic attack rate variation with age was estimated to be similar across four pandemics with very different mortality impact.
Conclusions
In all influenza pandemics of the 20th century, emergent viruses resembled those that had circulated previously within the lifespan of then-living people. Such individuals were relatively immune to the emergent strain, but this immunity waned with mutation of the emergent virus. An immune subpopulation complicates and may invalidate vaccine trials. Pandemic influenza does not 'shift' mortality to younger age groups; rather, the mortality level is reset by the virulence of the emerging virus and is moderated by immunity of past experience. In this study, we found that after immune escape, older age groups showed no further mortality reduction, despite their being the principal target of conventional influenza vaccines. Vaccines incorporating variants of pandemic viruses seem to provide little benefit to those previously immune. If attack rates truly are similar across pandemics, it must be the case that immunity to the pandemic virus does not prevent infection, but only mitigates the consequences.
doi:10.1186/1741-7015-10-162
PMCID: PMC3554498  PMID: 23234604
Pandemic influenza; mortality due to influenza; recycling; pandemic attack rates; vaccination; protective immunity
18.  Toll-Like Receptor 2 Mediates Fatal Immunopathology in Mice During Treatment of Secondary Pneumococcal Pneumonia Following Influenza 
The Journal of Infectious Diseases  2011;204(9):1358-1366.
Host inflammatory responses contribute to the significant immunopathology that occurs during treatment of secondary bacterial pneumonia following influenza. We undertook the present study to determine the mechanisms underlying disparate outcomes in a mouse model with β-lactam and macrolide antibiotics. Lysis of superinfecting bacteria by ampicillin caused an extensive influx of neutrophils into the lungs resulting in a consolidative pneumonia, necrotic lung damage, and significant mortality. This was mediated through Toll-like receptor (TLR) 2 and was independent of TLR4 and the Streptococcus pneumoniae cytotoxin pneumolysin. Treatment with azithromycin prevented neutrophil accumulation and rescued mice from subsequent mortality. This effect was independent of the antibacterial activity of this macrolide since dual therapy with ampicillin and azithromycin against an azithromycin-resistant strain also was able to cure secondary pneumonia. These data suggest that strategies for eliminating bacteria without lysis coupled with immunomodulation of inflammation should be pursued clinically.
doi:10.1093/infdis/jir522
PMCID: PMC3218647  PMID: 21900488
19.  Immunopathogenic and Antibacterial Effects of H3N2 Influenza A Virus PB1-F2 Map to Amino Acid Residues 62, 75, 79, and 82▿ 
Journal of Virology  2011;85(23):12324-12333.
The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation.
doi:10.1128/JVI.05872-11
PMCID: PMC3209399  PMID: 21937639
20.  Mathematical Model of a Three-Stage Innate Immune Response to a Pneumococcal Lung Infection 
Journal of theoretical biology  2011;276(1):106-116.
Pneumococcal pneumonia is a leading cause of death and a major source of human morbidity. The initial immune response plays a central role in determining the course and outcome of pneumococcal disease. We combine bacterial titer measurements from mice infected with Streptococcus pneumoniae with mathematical modeling to investigate the coordination of immune responses and the effects of initial inoculum on outcome. To evaluate the contributions of individual components, we systematically build a mathematical model from three subsystems that describe the succession of defensive cells in the lung: resident alveolar macrophages, neutrophils and monocyte-derived macrophages. The alveolar macrophage response, which can be modeled by a single differential equation, can by itself rapidly clear small initial numbers of pneumococci. Extending the model to include the neutrophil response required additional equations for recruitment cytokines and host cell status and damage. With these dynamics, two outcomes can be predicted: bacterial clearance or sustained bacterial growth. Finally, a model including monocyte-derived macrophage recruitment by neutrophils suggests that sustained bacterial growth is possible even in their presence. Our model quantifies the contributions of cytotoxicity and immune-mediated damage in pneumococcal pathogenesis.
doi:10.1016/j.jtbi.2011.01.052
PMCID: PMC3066295  PMID: 21300073
Bacterial Dynamics Model; Streptococcus pneumoniae Infection; Acute Inflammation; Dose-Dependence; Immune Response Modeling
21.  Fatal Outcome of Pandemic H1N1 2009 Influenza Virus Infection Is Associated with Immunopathology and Impaired Lung Repair, Not Enhanced Viral Burden, in Pregnant Mice ▿  
Journal of Virology  2011;85(21):11208-11219.
Pandemic A (H1N1) 2009 influenza virus (pH1N1) infection in pregnant women can be severe. The mechanisms that affect infection outcome in this population are not well understood. To address this, pregnant and nonpregnant BALB/c mice were inoculated with the wild-type pH1N1 strain A/California/04/09. To determine whether innate immune responses are associated with severe infection, we measured the innate cells trafficking into the lungs of pregnant versus nonpregnant animals. Increased infiltration of pulmonary neutrophils and macrophages strongly correlated with an elevated mortality in pregnant mice. In agreement with this, the product of nitric oxide (nitrite) and several cytokines associated with recruitment and/or function of these cells were increased in the lungs of pregnant animals. Surprisingly, increased mortality in pregnant mice was not associated with higher virus load because equivalent virus titers and immunohistochemical staining were observed in the nasal cavities or lungs of all mice. To determine whether exacerbated inflammatory responses and elevated cellularity resulted in lung injury, epithelial regeneration was measured. The lungs of pregnant mice exhibited reduced epithelial regeneration, suggesting impaired lung repair. Despite these immunologic alterations, pregnant animals demonstrated equivalent percentages of pulmonary influenza virus-specific CD8+ T lymphocytes, although they displayed elevated levels of T-regulator lymphocytes (Tregs) in the lung. Also, pregnant mice mounted equal antibody titers in response to virus or immunization with a monovalent inactivated pH1N1 A/California/07/09 vaccine. Therefore, immunopathology likely caused by elevated cellular recruitment is an implicated mechanism of severe pH1N1 infection in pregnant mice.
doi:10.1128/JVI.00654-11
PMCID: PMC3194964  PMID: 21865394
22.  Glycan Shielding of the Influenza Virus Hemagglutinin Contributes to Immunopathology in Mice 
Rationale: Pandemic influenza viruses historically have had few potential sites for N-linked glycosylation on the globular head of the hemagglutinin (HA) on emergence from the avian reservoir. Gain of glycans within antigenic sites of the HA during adaptation to the mammalian lung facilitates immune evasion.
Objectives: In this study, we sought to determine in mice how exposure to highly glycosylated viruses affects immunity to poorly glycosylated variants to model the emergence of a novel pandemic strain of a circulating subtype.
Methods: We engineered the 1968 H3N2 pandemic strain to express an additional two or four potential sites for glycosylation on the globular head of the HA. Mice were infected sequentially with highly glycosylated variants followed by poorly glycosylated variants and monitored for immune responses and disease.
Measurements and Main Results: The mutant with four additional glycosylation sites (+4 virus) elicited significantly lower antibody responses than the wild-type or +2 virus and was unable to elicit neutralizing antibodies. Mice infected with the +4 virus and then challenged with wild-type virus were not protected from infection and experienced significant T-cell–mediated immunopathology. Infection with a recent seasonal H1N1 virus followed by infection with the 2009 pandemic H1N1 elicited similar responses.
Conclusions: These data suggest that sequential infection with viral strains with different surface glycosylation can prime the host for immunopathology if a neutralizing antibody response matching the T-cell response is not present. This mechanism may have contributed to severe disease in young adults infected with the 2009 pandemic virus.
doi:10.1164/rccm.201007-1184OC
PMCID: PMC3159075  PMID: 20935106
influenza virus; glycosylation; pandemic; immunopathology; pneumonia
23.  Influenza Virus Primes Mice for Pneumonia From Staphylococcus aureus 
The Journal of Infectious Diseases  2011;203(6):880-888.
Superinfections from Staphylococcus aureus following influenza are an increasing concern. We assessed several laboratory and clinical strains in a mouse coinfection model with influenza virus. A methicillin-resistant USA300 clone and several recent clinical strains from patients with necrotizing pneumonia caused high mortality following influenza virus infection in mice. Both viral and bacterial lung titers were enhanced during coinfections compared with single infections. However, differences in titers did not correspond with differences in disease outcomes in a comparison of superinfections from a highly pathogenic strain with those from a poorly pathogenic strain. These strains did differ, however, in expression of Panton-Valentine leukocidin and in the degree of inflammatory lung damage each engendered. The viral cytotoxin PB1-F2 contributed to the negative outcomes. These data suggest that additional study of specific bacterial virulence factors involved in the pathogenesis of inflammation and lung damage during coinfections is needed.
doi:10.1093/infdis/jiq113
PMCID: PMC3071123  PMID: 21278211
24.  Influenza Enhances Susceptibility to Natural Acquisition and Disease from Streptococcus pneumoniae in Ferrets 
The Journal of infectious diseases  2010;202(8):1287-1295.
The role of respiratory viruses in transmission of Streptococcus pneumoniae is poorly understood. Key questions such as which serotypes are most fit for transmission and disease, and whether influenza virus alters these parameters in a serotype specific manner have not been adequately studied. In a novel model of ferret transmission, we demonstrated that prior infection with influenza virus of donors enhanced pneumococcal transmission and disease. Nasal wash bacterial titers, the incidence of mucosal and invasive disease, and the percentage of contacts infected were all increased. Viral infection of contact ferrets increased their susceptibility to acquisition both in terms of percentage infected and distance over which they could acquire infection. These influenza mediated effects on colonization, transmission and disease were pneumococcal strain dependent. Overall, these data argue that human studies of the relationship between respiratory viral infections, acquisition of pneumococci, and development of disease need further study to be better understood.
doi:10.1086/656333
PMCID: PMC3249639  PMID: 20822454
25.  Immunotherapy with a Combination of Intravenous Immune Globulin and P4 Peptide Rescues Mice from Postinfluenza Pneumococcal Pneumonia▿ 
Alternate therapies are needed for treatment of secondary bacterial pneumonia following influenza. The immunomodulatory peptide P4 has shown promise in mouse models of primary pneumococcal infection. Mice infected with influenza virus and then challenged with Streptococcus pneumoniae were treated with a combination of P4 peptide and intravenous immune globulin. Survival was improved from 20% to 80% in treated mice relative to controls. Clinical cure correlated with increased clearance of bacteria and decreased lung consolidation. Greater trafficking of professional phagocytic cells to the site of pneumococcal infection coupled with enhanced opsonophagocytosis as manifest by decreased surface display of Fcγ receptors (FcγR) on neutrophils and macrophages were associated with P4 peptide treatment. This suggests that the mechanism of action for improved clearance of bacteria engendered by P4 is through improved uptake by phagocytes mediated by IgG Fc-Fcγ receptor interactions following antibody-mediated opsonophagocytosis of bacteria. Antibody-based therapies, when coupled with immune modulators, such as P4 peptide, may be an effective tool together with antibiotics in our armamentarium against severe pneumonia.
doi:10.1128/AAC.00057-11
PMCID: PMC3088253  PMID: 21383090

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