A bacterial vaccine polypeptide protective against nontypable Haemophilus influenzae.

Nontypeable strains of Haemophilus influenzae (NTHi) are one of the most common cause of otitis media and the most frequent infection associated with exacerbations of chronic obstructive pulmonary disease; there is currently no vaccine in the U.S. to prevent NTHi. Using bioinformatics and structural vaccinology, we previously identified several NTHi species-conserved and sequence-conserved peptides that mediate passive protection in the rat model of infection. Using these, and similar peptides, we designed Hi Poly 1, a Bacterial Vaccine Polypeptide, comprising 9 unique peptides from 6 different surface proteins. Recombinant Hi Poly 1 was purified by affinity chromatography. Forty chinchillas were immunized three times with 200 µg of Hi Poly 1 with alum adjuvant; similarly, 41 controls were immunized with adjuvant alone. The average Log2 IgG titer among immunized animals was 17.04, and IgG antibodies against each component peptide were detected. In the infant rat model, antisera from immunized chinchillas provided significant passive protection compared to PBS (p = 0.01) and pre-immune sera (p = 0.03). In the established chinchilla model of NTHi otitis media, the vaccinated group cleared infection faster than the control group as indicated by significantly decreased positive findings on video-otoscopy (p < 0.0001) and tympanometry (p = 0.0002) on day 7, and for middle ear fluid obtained by aspiration (p = 0.0001) on day 10 post-infection. Using 12 representative NTHi strains in a Live-Cell ELISA, greater antibody binding to each strain was detected with post Hi Poly 1 than the pre-immune chinchilla antisera. The data from this proof-of-principle study demonstrate the effectiveness of Hi Poly 1 against the NTHi in two relevant preclinical models of bacteremia and otitis media as well as surface antibody binding across the species. The Bacterial Vaccine Polypeptide approach to a vaccine against NTHi also serves as a paradigm for development of similar vaccines to protect against other bacteria.

Targeted sequencing of candidate genes of dyslipidemia in Punjabi Sikhs: Population-specific rare variants in GCKR promote ectopic fat deposition.

Dyslipidemia is a well-established risk factor for cardiovascular diseases. Although, advances in genome-wide technologies have enabled the discovery of hundreds of genes associated with blood lipid phenotypes, most of the heritability remains unexplained. Here we performed targeted resequencing of 13 bona fide candidate genes of dyslipidemia to identify the underlying biological functions. We sequenced 940 Sikh subjects with extreme serum levels of hypertriglyceridemia (HTG) and 2,355 subjects were used for replication studies; all 3,295 participants were part of the Asian Indians Diabetic Heart Study. Gene-centric analysis revealed burden of variants for increasing HTG risk in GCKR (p = 2.1x10-5), LPL (p = 1.6x10-3) and MLXIPL (p = 1.6x10-2) genes. Of these, three missense and damaging variants within GCKR were further examined for functional consequences in vivo using a transgenic zebrafish model. All three mutations were South Asian population-specific and were largely absent in other multiethnic populations of Exome Aggregation Consortium. We built different transgenic models of human GCKR with and without mutations and analyzed the effects of dietary changes in vivo. Despite the short-term of feeding, profound phenotypic changes were apparent in hepatocyte histology and fat deposition associated with increased expression of GCKR in response to a high fat diet (HFD). Liver histology of the GCKRmut showed severe fatty metamorphosis which correlated with ~7 fold increase in the mRNA expression in the GCKRmut fish even in the absence of a high fat diet. These findings suggest that functionally disruptive GCKR variants not only increase the risk of HTG but may enhance ectopic lipid/fat storage defects in absence of obesity and HFD. To our knowledge, this is the first transgenic zebrafish model of a putative human disease gene built to accurately assess the influence of genetic changes and their phenotypic consequences in vivo.

Draft Genome Sequences of Eight Nontypeable Haemophilus influenzae Strains Previously Characterized Using an Electrophoretic Typing Scheme.

Nontypeable Haemophilus influenzae is an important cause of human disease. Strains were selected for genome sequencing to represent the breadth of nontypeable strains within the species, as previously defined by the electrophoretic mobility of 16 metabolic enzymes.

Draft Genome Sequences of Six Nontypeable Haemophilus influenzae Strains That Establish Bacteremia in the Infant Rat Model of Invasive Disease.

Haemophilus influenzae is an important cause of invasive disease. The infant rat is the accepted model of invasive H. influenzae disease. Here, we report the genome sequences of six nontypeable H. influenzae strains that establish bacteremia in the infant rat.

Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness.

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.

Prospective comparison of methicillin-susceptible and methicillin-resistant community-associated Staphylococcus aureus infections in hospitalized patients.

BACKGROUND: We sought to determine the proportion of community-associated Staphylococcus aureus infections due to methicillin-resistant S. aureus (CA-MRSA) at a large county hospital. In addition, we sought to identify the demographic and clinical risk factors associated with CA-MRSA infection. METHODS: Patients were prospectively enrolled if they were admitted to Parkland Hospital and had a positive culture for S. aureus isolated within 72 h of admission. The patients were interviewed using a standardized data questionnaire. Data collected included patient demographics, clinical history, as well as health care and non-health care associated MRSA risk factors. Bacterial susceptibilities were verified through review of microbiology laboratory and pharmacy records. Isolates were tested for Panton-Valentine leukocidin (PVL) gene, SCCmec type, and for inducible clindamycin resistance. RESULTS: One hundred and ninety-eight patients were interviewed prospectively, of which eight had colonization without active infection. One hundred and nineteen patients were infected with MRSA and 71 patients were infected with methicillin-susceptible S. aureus (MSSA). Patients with MRSA were more likely to be African-American and unemployed. Patients with MRSA most commonly presented with a skin or soft tissue infection (SSTI): 69% versus 45%, p=0.0012, while patients with MSSA were more likely to have infection of the respiratory tract: 11% versus 3%, p=0.02. Patients with MRSA were more likely to have used antibiotics in the past six months, been homeless, have a history of incarceration, have abused alcohol and have a history of infection with MRSA. In multivariate analysis, African-American race, antibiotics in the past six months, and a history of being homeless were associated with MRSA infection. Only 11 of 119 (9%) MRSA patients did not have at least one of these risk factors. PVL gene was present in 72 of 74 (97%) MRSA isolates and SCCmec type IV was present in 63 of 75 (84%) MRSA isolates. CONCLUSIONS: The majority of patients hospitalized with community-associated S. aureus infections were due to MRSA, most of which involved an SSTI. African-American race, recent antibiotics and past homeless status predicted infection with MRSA; however, no clinical profile could reliably exclude MRSA. Clinicians should be aware of the increasing prevalence of CA-MRSA.