Search

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.

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.

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.

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.

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.