Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspAbased vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

BACKGROUND Secondary bacterial infections after influenza can be a serious problem, especially in young children and the elderly, yet the efficacy of current vaccines is limited. Earlier work demonstrated that a replication-incompetent PB2-knockout (PB2-KO) influenza virus possessing a foreign gene in the coding region of its PB2 segment can serve as a platform for a bivalent vaccine. METHODS In the current study, we generated the PB2-KO virus expressing pneumococcal surface protein A (PspA), PB2-KO-PspA virus, the replication of which is restricted to PB2-expressing cells. We then examined the protective efficacy of intranasal immunization with this virus as a bivalent vaccine in a mouse model. RESULTS High levels of influenza virus-specific and PspA-specific antibodies were induced in the serum and airways of immunized mice. The intranasally immunized mice were protected from lethal doses of influenza virus or Streptococcus pneumoniae. These mice were also completely protected from secondary pneumococcal pneumonia after influenza virus infection. CONCLUSIONS These findings indicate that our recombinant influenza virus serves as a novel and powerful bivalent vaccine against primary and secondary pneumococcal pneumonia as well as influenza.

An increase in the appearance of nonvaccine serotypes in both children and adults with invasive pneumococcal disease (IPD) after introduction of pneumococcal conjugate vaccine represents a limitation of this vaccine. In this study, we generated three recombinant pneumococcal surface protein A (PspA) proteins comprising PspA families 1 and 2, and we examined the reactivity of antisera raised in mice immunized with a PspA fusion protein in combination with CpG oligonucleotides plus aluminum hydroxide gel. The protective effects of immunization with PspA fusion proteins against pneumococcal challenge by strains with five different PspA clades were also examined in mice. Flow cytometry demonstrated that PspA3+2-induced antiserum showed the greatest binding of PspA-specific IgG to all five challenge strains with different clades. PspA2+4- or PspA2+5-induced antiserum showed the lowest binding of PspA-specific IgG to clade 3. Immunization with PspA3+2 afforded significant protection against pneumococcal challenge by five strains with different clades in mice, but immunization with PspA2+4 or PspA2+5 failed to protect mice from pneumococcal challenge by strains with clades 1 and 3. The binding of PspA-specific IgG in antisera raised by three PspA fusion proteins was examined in 68 clinical isolates from adult patients with IPD. Immunization of mice with PspA3+2-induced antiserum with a high binding capacity for clinical isolates expressing clades 1-4, but not clade 5. Our results suggest that the PspA3+2 vaccine has an advantage over the PspA2+4 or PspA2+5 vaccine in terms of a broad range of cross-reactivity with clinical isolates and cross-protection against pneumococcal challenge in mice.

ABSTRACT Streptococcus pneumoniae is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases. IMPORTANCE Streptococcus pneumoniae and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or Streptococcus pneumoniae. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform for the development of bivalent vaccines.

8 Depertment of Special Pathogens, International Research Center for Infectious Diseases, Institute of 20 Medical Science, University of Tokyo, Tokyo, Japan 21 *These authors contributed equally to this work. 22 23 †,‡ Corresponding authors and mailing addresses: 24 †Kazunori Oishi 25 JVI Accepts, published online ahead of print on 10 September 2014 J. Virol. doi:10.1128/JVI.01205-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37 °C. In particular, intracellular growth in amoebae was completely impaired at 25 °C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37 °C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37 °C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.

To develop a cost-effective pneumococcal vaccine, we compared the effects of a panel of Toll-like receptor (TLR) agonists on a low dose pneumococcal surface protein A (PspA) nasal vaccine in a fatal pneumococcal pneumonia model using a serotype 3 strain. The mice were nasally immunized with 10 µg of the TLR agonist (TLR 2, 3, 4 and 9) and 0.1µg of PspA once per week for three weeks. A high level of PspA-specific immunoglobulin G (IgG) was detected in sera of mice that were nasally administered a low dose of PspA plus each TLR agonist, while no PspAspecific IgG were detected in sera of mice that had been nasally administered a low dose of PspA. A relatively low level of PspA-specific IgG was also detected in the airway of mice that had been nasally administered a low dose of PspA plus each TLR agonist. The binding of PspA-specific IgG increased the deposition of C3 on the bacterial surface. Bacterial density in the lung and blood was significantly decreased in mice that had been administered a low dose of PspA plus each TLR agonist, compared with mice that received a low dose of PspA alone 24 h after a bacterial challenge. Furthermore, significant increases in survival rate were found in a murine model of fatal pneumonia that had been nasally administered a low dose of PspA plus each TLR agonist, compared with mice that received a low dose of PspA alone. The rank order of TLR agonists on the effect of increasing survival rate was LPS > Pam3CSK4 > Poly (I:C) and CpG 1826. These data suggest a potentially new strategy for the development of a cost-effective intranasal vaccine with a low dose PspA plus TLR agonist that would be effective against lifethreatening bacteremic pneumococcal pneumonia.

Effective pneumococcal vaccines are required for preventing secondary bacterial pneumonia, a life-threatening condition, during epidemics of influenza. We examined whether nasal administration of a low dose of pneumococcal surface protein A (PspA) plus polyinosinic–polycytidylic acid (poly(I:C)) could protect against a fatal secondary pneumococcal pneumonia after influenza A virus infection in mice. PspA-specific IgG but not IgA level was higher in the airways and blood of mice nasally administered a low dose of PspA plus poly(I:C) than in mice nasally administered PspA alone or poly(I:C) alone. Binding of PspA-specific IgG increased C3 deposition on the bacterial surface. The survival rate during secondary infection was higher in mice immunized with PspA plus poly(I:C) than in mice immunized with poly(I:C) alone. The significant reduction in bacterial density in the lung and blood was associated with increased survival of immunized mice with secondary pneumonia. Passive transfer of sera from mice immunized with PspA plus poly(I:C) increased the survival of mice infected with secondary pneumonia. Our data suggest that an intranasal PspA vaccine has promising protective effects against secondary pneumonia after influenza and that PspA-specific IgG plays a critical role in this protection.

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.

Dual fuel natural gas engines have been studied for the purpose of near future engine. This study focuses on the effect of early injection of light oil and exhaust gas recirculation (EGR) fueled with methane from an inlet pipe on characteristics of combustion and exhaust emissions. The injection timing was changed from TDC to 50 degrees before the TDC. In the early injection timing, smoke was never seen and hydrocarbons were smaller compared with those at the normal injection timing. However, the combustion becomes too early to obtain an appropriate torque when the equivalence ratio increases. Then, moderate nitrogen dilution was very effective to retard the combustion with lower NOX, higher thermal efficiency and almost the same hydrocarbons and carbon monoxide. And it was found that the engine operates even under the condition of stoichiometric mixture.

In diesel engines, there is a problem that smoke and oxide of nitrogen cannot be reduced simultaneously. There have been many studies on solving this problem. One of the solutions is an engine that uses gaseous fuel. Gaseous fuel is inducted from an intake port and the combustion starts by injecting light oil. And hydrogen is considered to be future fuel without exhausting carbon dioxide. In this study, hydrogen was inducted from intake port into the cylinder of a diesel engine and light oil was injected in the cylinder. The heat release rate and the exhaust emissions are discussed. As a result, when hydrogen is mixed with the inlet air, exhaust emissions such as smoke, hydrocarbon, CO, CO_2, and NO_x are reduced simultaneously though brake thermal efficiency is slightly low.

In diesel engines, there is a problem that both smoke and nitrogen oxide cannot be reduced simultaneously. There have been many studies on solving this problem. One of the solutions is utilization of gaseous fuel. Gaseous fuel is inducted from an intake port and the combustion starts by injecting light oil. Hydrogen is considered as a future fuel without exhausting carbon dioxide. In this study, hydrogen was inducted from intake port into the cylinder of a diesel engine and light oil was injected in the cylinder. The heat release rate was determined from the pressure history in the cylinder. The relation between the heat release rate and the exhaust emissions are discussed. As a result, when hydrogen is mixed with the inlet air, exhaust emissions such as smoke, hydrocarbon, CO, CO2, and NOx are reduced simultaneously while brake thermal efficiency is slightly lower.

In diesel engines, ignition delay is determined as the period between fuel injection and ignition. The ignition delay affects the initial combustion and the following combustion process. In this study, the gas fuel (propane, methane or hydrogen) mixed with air was induced into the cylinder and the light oil was injected as ignition source. Here, the ignition delay in this kind of engine was focused as a basic study. Even in the dual fuel like this engine, the ignition delay time is predicted by changing the value of the constant, A in Livengood-Wu equation while the others constants is the same in an ordinary diesel engine. Ignition delay becomes long in air/methane or air/propane mixture because the temperature at injection timing of light oil decreases. In air/hydrogen mixture, the ratio of specific heats increases slightly, however, the mole fraction of oxygen decreases due to decrease of the mass of air. Therefore, the ignition delay in air/hydrogen mixture is a little longer than that in just air. When the light oil is injected near at top dead center, slight heat release appears due to the self-ignition of gas fuel and the cycle-to-cycle variations of the ignition delay and the following combustion increase.