Projects Ongoing Projects Identification of host responses to Respiratory Syncytial Virus (RSV) Infection and Identification of factors associated with severe disease (Walsh) A clinical translational study in two cohorts of infants, representing the full spectrum of RSV disease severity, in which innate and adaptive immune status are comprehensively measured in association with environmental, viral, and bacteriologic factors. Impact of respiratory virus infections and bacterial microbiome shifts on lymphocyte (Lc) and respiratory function in infants born prematurely or full term (Pryhuber) A clinical research study to investigate the relationships between sequential respiratory viral infections, patterns of intestinal and respiratory bacterial colonization, and adaptive cellular immune phenotypes which are associated with increased susceptibility to respiratory infections and long term respiratory morbidity in preterm and full term infants. Innovation Projects Validation of Gene Array to Predict Bacterial Co-infection In Adults Hospitalized with Viral Lower Respiratory Tract Infections (LRTI) (Mariani/Fasley) The primary goal of the current innovation project is to conduct analysis of the RNASeq data collected in DMID 13-0070, in an effort to identify gene expression variables capable of classifying, or predicting, bacterial and viral infection in adults with LRTI. The secondary goal is to perform gene expression validation of previously identified and new predictors. Serum Biomarkers associated with bacterial respiratory infections in hospitalized adults (Falsey) This project will utilize serum banked from prior NIH funded project 1R01AI079446-01 “Reduction of Unnecessary Antibiotics in Adults by the Use of Viral Diagnostics” In this study, blood was prospectively collected at time of admission and 4 weeks after illness resolution from subjects hospitalized with respiratory illness. Each illness had extensive microbiologic testing performed in order to categorize illnesses as bacterial, viral or mixed bacterial and viral. The proposed project will utilize these banked sera and test the utility of 3 newly identified serum biomarkers to differentiate bacterial and viral respiratory infection. Informed consent was obtained in the original grant to test serum biomarkers for this purpose. Testing of Sputum by Film Array for viral and atypical bacterial respiratory pathogens (Falsey) The purpose of this project was to develop a simple method to test sputum in a fully automated PCR system. Archived samples from R01AI079446-01 were used. The work was consistent with the original intent and consent of the study to develop better tests for diagnosis of viral and bacterial infection. Testing for an impaired function of cord blood T regulatory cells from preterm neonates who are predisposed to developing severe respiratory viral infection versus gestational age-matched controls (Misra) The overall goal of the project is to provide a manuscript at the end of the study period, which describes the function of T regulatory cells from the cord blood of infants who are born preterm in the presence of inflammation of the umbilical cord and those who develop the lung disease Bronchopulomnary Dysplasia. Pneumococcal Colonization in Older Adults (Falsey) Determine the cumulative incidence of pneumococcal carriage over an 8 month period of time in persons > 65 years of age. Evaluate potential variables that influence the risk of carriage. Depending on the cumulative incidence the following secondary objectives will be explored. Samples will be collected and stored in such a way to allow future testing as warranted. Novel in vivo methodologies for the characterization of potential protective antigens against respiratory infection with Bordetella pertussis (Hewlett/Damron) In this project, we will use a novel mouse model of infection to test adenylate cyclase toxin (ACT) as a vaccine antigen. These trials will test ACT alone and in combination with currently used acellular pertussis vaccines. The mouse strain, known as NeCre luc, has bioluminescent neutrophils, which allow us to analyze the immune response to pertussis infection. Vaccines that promote neutralization of B. pertussis through an efficient immune response would be the preferred candidates for further studies and in the future, clinical trials. H5 Influenza Vaccine Study: Gene Expression and HAI Antibody Response Analysis (Topham) This study takes advantage of existing clinical samples (frozen PBMC) collected as part of DMID-08-0059. This study was comprised of three groups of subjects that differ in their pre-vaccination immune status. All subjects are healthy adults between the ages of 18 and 64. Samples for assessment of B cell and CD4 T cell responses were obtained on days 0, 3, 7, 14, and 28 following each dose of vaccine. In the current project, we plan to use the day 0, 3 and 7 samples after each immunization to assess host gene expression. The goal is to better understand differences in the innate immune response to vaccination that predict vaccine outcome (production of antibody). Bacterial Respiratory Pathogen Reference Laboratory (Nahm) The Analytical Support Laboratory will perform various serological tasks required by the NIH for the Respiratory Pathogen Research Center (RPRC) at the University of Rochester. The tasks include 1) Have capacity to analyze 500 sera samples per year for pneumococcal antibodies by MOPA (multiplexed opsonophagocytosis assay), 2) Have capacity to analyze 500 sera samples per year for pneumococcal antibodies by ELISA, 3) Have capacity to precisely serotype 100 S. pneumoniae isolates, 4) Support development of an opsonophagocytosis assay for antibodies to Group A streptococcus, 5) If needed, teach the above methods to visitors from other labs (two to four visitors per year), 6) Continue standardization and validation of MOPA for pneumococcal antibodies, 7) Continue developing an assay to identify serotypes causing pneumococcal pneumonia, and 8) performing MOPA for samples from DMID protocol 11-0034. Effect of age on T cell responses to Respiratory Syncytial Virus infection in Adults. (Walsh) This one year innovation project will utilize archived PBMCs collected during a prior DMID study (05-0073) from young and older person with RSV infection, and from a group of non-infected adults of matched age. We will analyze T cell responses after stimulation with RSV in three groups of subjects. Groups include young persons (<40 years of age) with mild disease, older persons (>65 years of age) and older persons with severe RSV disease. We hypothesize that older persons with mild disease will have lower Th1 and Treg responses and greater Th2 responses compared to younger persons with mild disease. We further hypothesize that older persons with severe disease will have greater Th2 and diminished Th1 responses compared to older persons with mild disease. T cell responses will be assayed using multicolor flow cytometry after stimulation with RSV antigens. Studies of human memory B cells: optimization of analytical strategies and characterization of influenza H7 hemagglutinin-reactive memory B cells induced by vaccination (Sangster) The characteristics of an individual’s memory B cell population are a key determinant of susceptibility to influenza-induced disease and responsiveness to influenza vaccination, but there is little agreement on optimal and standardized strategies for memory B cell analysis. This project will evaluate and develop strategies to measure the frequencies of antigen-specific memory B cells. Optimized approaches will be applied to evaluate the response to human influenza vaccination and the induction of memory B cells reactive with the H7 HA of emerging influenza viruses with pandemic potential. Cell-mediated and Humoral Antibody Subclass Assay Development for Immune profiling of response in human subjects to Whole cell and Acellular Pertussis vaccines after priming and boosting. (Quataert) Infections with Bordetella Pertussis are increasing in the U.S. since the replacement of whole cell Pertussis (wP) vaccines with acellular Pertussis (aP) vaccines over the last two decades (CDC surveillance reports). While antibody responses to both the wP and aP vaccines appear to be similar in magnitude for some of the main shared components (PT, FHA, PRN and or FMI 2&3) as measured by ELISA, the type and duration of antibody differ. To effectively design and test new Pertussis vaccine formulations, standardized, validated assay methods are important for mechanistically measuring what characterizes a “protective” response generating long-term memory. We propose to develop assays to characterize and monitor CD4+ T cell responses in different types of priming and boosting, to design strategies to increase the effectiveness of current vaccines, and to help in the design and evaluation of more efficacious new Pertussis vaccines. We will also standardize and validate new multiplexed antibody assays for use in evaluating pertussis vaccines as well as establish weight-based assignments for subclass antibodies allowing better assessment of protective levels for different subclass responses and correlation to other CMI and functional assays. ß-lactam antibiotic resistance in Mycobacterium abscessus (Pavelka) We will determine the susceptibility of M. abscessus strains to various β-lactam antibiotics in the presence or absence of different β-lactamase inhibitors. We will also clone the two β-lactamase genes from M. abscessus into a plasmid for overexpression in E. coli for purification, followed by in vitro β-lactamase enzyme assays. Finally, we will construct mutant strains of M. abscessus and evaluate their antibiotic resistance profiles. A Multiplex Label-Free Chip for Pneumococcus Serology (Miller/Nahm) We will develop a new label-free antibody microarray for Pneumoccus serology. As part of this process, we will benchmark the performance of the new array versus the standard serologic methodology using bacterial culture supernatants. Completion of these initial studies will set the stage for an expanded development effort targeting a pneumococcus serology array incorporating antibodies to all known serotypes. Impact of Influenza-specific Regulatory T cells on the Magnitude of Immunity of Influenza Infection and Vaccination (Fowell) Regulatory T cells (Tregs) play a fundamental role in modulating immune responses: homeostatically to self-antigen and following infectious challenge. Originally shown to dampen immune responses to infection and limit pathogen clearance, in recent years we have begun to appreciate the beneficial role of Tregs in limiting immune pathology associated with infection. Therefore, the balance between detrimental and beneficial roles for Tregs is likely to be context dependent and pathogen specific. Critical gaps in knowledge of Tregs in influenza: CD4+Foxp3+ Tregs expand to influenza infection in mice with similar kinetics to conventional CD4+ T cells (Tconv) and some of these expanded Tregs appear to be virus-specific. Using TCR transgenic systems, antigen-specific Tregs can be shown to limit the recall response to influenza infection and control the extent of infection-induced immunopathology. However, in the absence of elevated frequencies afforded by a TCR-Tg, the role of virus-specific Tregs in shaping responses to influenza is unclear. In humans, the degree of influenza-specific Treg expansion to infection or vaccination is not known. Nor do we know if repeated exposure to influenza antigens builds a robust virus-specific Treg population that modifies responsiveness to new viral challenge. Validation of Gene Array to predict bacterial co-infection in adults hospitalized with LRTI (Falsey) The objective of this project is to prospectively validate 15 classifier genes previously identified as useful to differentiate viral, bacterial and mixed viral- bacterial infections in adults hospitalized with LRTI. RNA sequencing will be used to validate previous findings and explore potentially new alternative gene expression patterns. To accomplish this goal ~ 300 adults hospitalized with LRTI will be prospectively enrolled, microbiologic testing performed and RNA sequencing will be performed in those with identifiable viral and or bacterial infection. Complement dependent lytic (CDL) and antibody dependent cellular cytotoxicity (ADCC) antibodies after influenza vaccination (Ennis) Antibodies neutralize influenza infectivity by binding to the HA protein or by inhibiting virus release by binding to the neuraminidase protein. In addition, influenza -specific antibodies can bind to infected cells, and lyse these cells through the action of complement (complement dependent lysis- CDL) or through NK cells (antibody dependent cellular cytotoxicity-ADCC). We are planning to test serum samples collected as part of experimental human H7N9 vaccine trials in order to better understand the role that CDL and ADCC antibodies play in protection against influenza infection. Development of an opsonophagocytosis assay for Group A streptococcus antibodies (Nahm) The Nahm laboratory has revolutionized pneumococcal vaccine evaluations by developing a practical and multiplexed opsonophagocytosis assays for pneumococcal antibodies. The Nahm laboratory is now developing an assay to measure opsonic capacity of antibodies induced with a group A streptococcal vaccine to facilitate the vaccine development. The vaccine is being developed by Dr. J. Dale in the University of Tennessee in Memphis. Multiplexed Immunoassays for Pneumococcal Polysaccharide (PnPs) Vaccine Evaluation (Quataert) New multiplexed imaging flow cytometry and bead array immunoassays applied to the assessment of clinical vaccine studies have great potential for accelerating the development and licensure of new vaccines. In this project, we propose to develop and qualify (pre-study validation) multiplexed flow-based imaging opsonphagocytic assays (OPA) and anti-PnPs isotype and subclass microbead array assays for use in evaluation of new or current PnPs vaccines against Streptococcus pneumoniae. Modeling HA Evolution (Scheuermann) Twice each year the WHO assembles a group of influenza experts to select virus strain candidates for vaccine development. Strains isolated toward the end of the previous influenza season are often selected based on the hypothesis that these represent strains escaping population immunity to the current seasonal flu and will likely emerge as the predominant strain in the next flu season. However, an explicit model that incorporates predictions about viral evolution in the face of immune system pressure has not been formally included. We propose to develop a model of influenza virus evolution that explicitly incorporates parameters derived from a genome-wide analysis of sequence variability and an analysis of protein regions with immune reactivity. Enhancement of anti-pertussis immune responses by bordetella colonization factor A (BcfA) (Deora) Despite high vaccine coverage, the incidence of pertussis is increasing in the USA, Europe and other developed countries. While current acellular pertussis vaccines (aPV) protect against the severe disease, they do not protect against infection and subsequent familial and individual-individual transmission. The lower efficacy of current aPVs is suggested as a major reason for the re-emergence of this disease. Effective immune protection to Bordetella pertussis is correlated with the generation of strong Th1 and Th17 responses following either infection with B. pertussis or immunization with whole cell vaccines (wPVs). However, the current aPVs produce mixed Th1/Th2 or more Th2-skewed responses. Alum, the current adjuvant in aPV fails to elicit appropriate immune responses for optimum protection against B. pertussis. Thus, substitution of alum with an adjuvant that induces Th1-type responses may increase vaccine efficacy. This could be particularly advantageous if the immune-stimulatory activity was derived from B. pertussis itself, thus providing both adjuvant function and an additional B. pertussis antigen in a novel aPV combination. We identified Bordetella Colonization Factor A (BcfA) protein in B. bronchiseptica and B. pertussis as such an immune-stimulatory factor. Our objective for this proposal is to incorporate BcfA into a new aPV and define the ability of BcfA to enhance immune responses to pertussis antigens. Combining mutagenesis, sequencing technologies and in vivo imaging to identify B. pertussis virulence factors and vaccine antigens (Eby) In the 1990’s, the acellular pertussis vaccine replaced the whole cell pertussis vaccine, and over the following ~20 years, there has been a 10-fold increase in the incidence of whooping cough or pertussis. In this project, we will use complementary genetic techniques to identify novel factors required for B. pertussis to establish infection. The tools produced in this project can be employed by the research community and the antigens identified may be evaluated as vaccine antigens. First, we will use in vivo RNA-seq to determine which genes are turned on/off during infection of mice with B. pertussis. Next, we will use in vivo transposon-seq to determine which genes contribute to the survival or fitness of B. pertussis during infection. After analyzing the data from these experiments in combination, selected genes will be eliminated from B. pertussis and the ability of these strains to infect mice measured by in vivo imaging. A Phase I randomized study in healthy adults to assess the safety, reactogenicity and immunogenicity of priming with an inactivated A/H7N9 influenza virus vaccine with or without MF59 Adjuvant followed by live attenuated A/H7N9 influenza virus vaccine (Treanor) To assess the safety of administration of H7N9 pLAIV administered to individuals who have previously received MF59-adjuvanted or unadjuvanted H7N9 pIIV.To evaluate the ability of a single dose of unadjuvanted H7N9 pIIV to prime for enhanced immunogenicity manifest as higher titer antibody response (booster response) to subsequent administration of antigenically-matched pLAIV H7N9 vaccine compared with subjects vaccinated with a single dose of MF59-adjuvanted H7N9 pIIV or a single dose of unadjuvanted H7N9 pIIV. To evaluate the ability of a single dose of MF59-adjuvanted H7N9 pIIV to prime for enhanced immunogenicity manifest as higher titer antibody response (booster response) to subsequent administration of antigenically-matched pLAIV H7N9 vaccine compared with subjects vaccinated with a single dose of MF59-adjuvanted H7N9 pIIV or a single dose of unadjuvanted H7N9 pIIV. Risk Factors and immune response to Pneumococcal Colonization in Older Adults (Branche) Primary Objectives:To determine the cumulative incidence, density and duration of pneumococcal carriage in older adults over a 12 month period as well as assess potential factors which may influence the risk of carriage with a focus on vaccination status. Compare rates of carriage in adults over age 65 who have received pneumococcal vaccine to adults age 55-64 who have not had any type of pneumococcal vaccination. Determine the effect of viral co-infections on the rate of colonization. Measure and compare baseline serum antibody levels in colonized vs. non colonized subjects and evaluate the serotype specific immune response to colonization. Determine the titer of bacteria present in NPS of adults found to be colonized with pneumococcus and compare titers in NPS of adults hospitalized with community acquired pneumonia. Improved diagnosis of respiratory infections using host gene expression (Storch) The broad objective of this proposal is to develop improved diagnostic tests that would empower physicians to limit antibiotic use in patients with symptoms of upper respiratory tract infection to those who truly have bacterial infection. This study will focus on two sites of infection – the nose and the oropharynx. The nose will be a critical site for obtaining diagnostic specimens for diagnosis of common upper respiratory tract infections. The oropharynx will be used because it is a site where bacterial and viral infections are common and can be accurately classified using existing tests, making it an ideal site for evaluating the new approach of host-based diagnosis. Genetic Determinants of Neurovirulence in the Recent Enterovirus D68 Outbreak (Scheuermann) The objective for this innovation project is to test the hypotheses that one or more of the unique substitutions identified in the recent EV-D68 dominant B.2 cluster have enhanced the ability of EV-D68 to replicate in neuronal cells either by affecting 1) the structure and function of the internal ribosomal entry site (IRES) in the 5’-UTR, 2) the efficiency of genome replication by affecting the function of RDRP encoded by the 3D protein, and/or 3) neuronal receptor specificity dictated by the VP1/VP2/VP3 capsid proteins, thereby identifying important diagnostic and therapeutic targets. Completed Projects Specificity and Fate of follicular helper T cells (Sant) A research project to analyze the specificity, distribution in vivo, and mobilization of Tfh cells using mouse models of vaccination and infection. The aims of this project are to determine the specificity of follicular helper cells elicited during protein vaccination or infection and analyze Tfh fate and recruitment to subsequent responses after influenza virus challenge. Flu Chip phototonic based antibody sensors to detect influenza antibodies (Miller) The Miller laboratory has developed lab-on-a-chip methods for influenza serology. These devices simplify profiling antibody responses to panels of influenza antigens, for applications in surveillance and vaccine development. A Multiplex Label-Free Chip for Pneumococcus Serology (Miller/Nahm) The goal of our project was to develop a simple, label-free, multiplex assay for pneumococcal serotyping, using the Arrayed Imaging Reflectometry (AIR) platform. As a proof of concept, antibodies specific to 16 serotypes will be used in combination with appropriate positive and negative controls to produce an antibody microarray. Following assessment of antibody specificity in the microarray context using purified capsular polysaccharides, bacterial culture supernatants will be used to benchmark the performance of the AIR array against standard serology methods. Randomized Clinical Trial to use viral testing and serum biomarkers to reduced unnecessary antibiotic use for respiratory infections (Falsey) The background for this small project was based on: the specific microbiologic diagnosis of lower respiratory tract infections remains problematic; PCR has markedly improved diagnosis of viral infection; bacterial diagnostics continue to be hampered by poor sensitivity and specificity; because it is difficult to “rule out” bacterial infection even in patients with documented viral illnesses, most patients receive broad spectrum antibiotics; antibiotic over use is a major public health issue; and novel methods to diagnose bacterial respiratory infections are urgently needed. The primary objectives for the project were to prospectively validate the 15 classifier genes previously identified as useful to differentiate viral, bacterial and mixed viral- bacterial infections in adults hospitalized with LRTI as well as perform RNA sequencing to validate previous findings and explore potentially new alternative gene expression patterns.