A universal influenza candidate vaccine that targets multiple conserved influenza virus epitopes from hemagglutinin (HA), neuraminidase (NA) and matrix (M2e) proteins was combined with the potent Army liposomal adjuvant (ALFQ) to promote induction of broad immunity to seasonal and pandemic influenza strains. The unconjugated and CRM-conjugated composite peptides formulated with ALFQ were highly immunogenic and induced both humoral and cellular immune responses in mice. Broadly reactive serum antibodies were induced across various IgG isotypes. Mice immunized with the unconjugated composite peptide developed antibody responses earlier than mice immunized with conjugated peptides, and the IgG antibodies were broadly reactive and neutralizing across Groups 1 and 2 influenza viruses. Multi-epitope unconjugated influenza composite peptides formulated with ALFQ provide a novel strategy for the development of a universal influenza vaccine. These synthetic peptide vaccines avoid the pitfalls of egg-produced influenza vaccines and production can be scaled up rapidly and economically.

studied. Aerosol therapy in mice is problematic due to the high cost of the required apparatus. The purpose of this study was to explore the efficacy of intranasal (IN) administration (as a surrogate for aerosol delivery) of isoniazid (INH) and rifalazil (RZL) in a murine tuberculosis model compared to oral delivery.Methods & Materials: Six week old female Balb/c mice (purchased from Charles River Laboratories, Wilmington, MA) were infected intranasally with about 10 3 CFU of Mycobacterium tuberculosis (MTB) ATCC 27294 (H37Rv) for experiment 1 or about 10 6 CFU of MTB ATCC 35801 (Erdman) for experiment 2. One week post infection mice in experiment 1 were treated with INH 5mg/kg orally by gavage or 5mg/kg IN for 3 days and mice in experiment 2 were treated with RZL 5 mg/kg orally by gavage 5 days/week or 5 mg/kg IN Mon, Wed, and Fri for 2 weeks. At the initiation of therapy in each experiment a group of mice (early controls) were euthanized by CO 2 inhalation and their lungs were collected. At the completion of therapy an untreated group of mice, late controls (LC), and treated mice were euthanized. Mycobacterial loads in right lungs were measured by serial dilution and plating on Middlebrook 7H10 agar plates.Results: The mycobacterial loads (log CFU) for the EC, LC, INH oral and INH IN were 3.34, 4.49, 2.94, and 2.82 respectively (exp. 1). The mycobacterial loads for the EC, LC, RZL oral, and RZL IN were 6.26, 8.93, 4.30, and 4.58 respectively (exp. 2). The LC group was euthanized 3 days early due to their advanced illness. IN delivery of INH and RIF was significantly better than the untreated late controls. Conclusion: The activities of INH and RZL by IN delivery in these experiments suggest that other agents that cannot be given orally could be evaluated for their potential therapeutic activities by IN administration. http://dx.Background: Mycobacterium tuberculosis (MTB) is an important cause of bacteremia and sepsis in HIV patients residing in sub-Saharan Africa. Many patients with MTB sepsis go undiagnosed and die within 18 days of presentation, making culture inadequate for detecting MTB in the blood. Objective:To determine the feasibility of ambient temperature transport of blood in PrimeStore MTM ® to a distant lab for real-time PCR detection of MTB bacteremia and to monitor clearance of MTB from the blood after therapy.Methods & Materials: BALB/c female mice were injected intravenously with 0.2 mls of ethanol killed MTB (approximately 10 5

BackgroundPatients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity.MethodsBALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells. Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN.ResultsTwo MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent.ConclusionsAnti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.

Lysostaphin is being developed as a treatment for serious staphylococcal infections. Mice challenged with S. aureus produce inflammatory cytokines including, TNF-and IL-6, and over-production of these cytokines can lead to shock and contribute to the lethality of staphylococcal infections. Two major components of the staphylococcal cell wall, peptidoglycan and lipoteichoic acid, are known to synergize to induce shock and organ failure in animal models, and we wished to determine whether the rapid lysostaphin-mediated degradation of peptidoglycan during treatment of systemic S. aureus infection could affect shock-associated parameters. We found that lysostaphin treatment of S. aureus-infected mice, which reduces bacteremia and organ infection, also reduced the serum levels of inflammatory cytokines and reversed the symptoms of S. aureus-induced shock. We compared the cytokine response of mice challenged with S. aureus to that of mice challenged with S. aureus and then treated with lysostaphin or nafcillin. Lysostaphin-treated mice, as compared with untreated mice or nafcillin-treated mice, had a blunted cytokine responses to S. aureus challenge. Core body temperature was used as a real time indicator for systemic shock in mice. Mice infected with S. aureus demonstrated a rapid drop in core body temperature, which was reversed by lysostaphin treatment. These studies demonstrated that lysostaphin treatment did not contribute to the induction of shock by rapidly releasing staphylococcal cell wall components, but rather it blunted the inflammatory cytokine response and ameliorated shock related symptoms.

The ability to measure the opsonic activity of antibody may be critical in choosing among potential therapeutic candidates. It has been shown in numerous studies that the ability of antibody to mediate clearance of bacterial organisms in animal models and in humans is related to its in vitro opsonic activity and not solely to its antigen binding activity. We have developed a rapid and robot adaptable opsonophagocytosis assay for Staphylococci based on bioluminescence produced by bacteria transfected with the luciferase operon. Opsonophagocytosis is measured by the loss of bioluminescence as bacteria are opsonized by phagocytes and killed. Pagibaximab is a humanized monoclonal anti-lipoteichoic acid (LTA) antibody opsonic for S. epidermidis and S. aureus, which we used to develop this rapid bioluminescence assay.