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. 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. The natural history of pulmonary nontuberculous mycobacterial (NTM) disease; a longitudinal population-based outcomes assessment of HIV-negative patients with pulmonary NTM (Winthrop) Nontuberculous mycobacterium (NTM) are emerging as an important cause of chronic pulmonary disease in older adults. Using a large population-based cohort, this project seeks to define the natural history, epidemiology, and outcomes of pulmonary NTM disease. 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. 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. 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. 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. 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. 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. 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. 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. 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. Innovation Projects 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. 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 is developing lab-on-a-chip methods for influenza serology. These devices will simplify profiling antibody responses to panels of influenza antigens, for applications in surveillance and vaccine development.