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Although many influenza-related deaths are attributable to secondary bacterial infection with S. pneumoniae, vaccines that simultaneously protect against influenza and pneumococcal infection are currently not developed. The aim of our study was to evaluate the possibility to prevent post-influenza pneumococcal infection using an associated vaccine based on live influenza vaccine (LAIV) combined with recombinant polypeptides derived from superficial factors of Group B streptococcus (GBS) determining pathogenicity. We demonstrated in a model of post-influenza pneumococcal pneumonia that intranasal pneumococcal super-infection seriously complicated the course of A/Shanghai/2/2013(H7N9) CDC-RG virus infection in mice. Associated immunization using LAIV and GBS vaccine (GBSV) prevented post-influenza pneumococcal pneumonia better than mono-LAIV or GBSV immunization. At the same time, parenteral pneumococcal post-influenza infection of immune mice was more severe in the groups immunized using recombinant GBS peptides which can be explained by antibody-dependent enhancement of infection. In this case, the introduction of blockers of histamine receptors type 1 and 2 reduced the burden of secondary pneumococcal infection., Competing Interests: The authors have declared that no competing interests exist.

We investigate the protective effect of combined vaccination based on live attenuated influenza vaccine (LAIV) and group B streptococcus (GBS) recombinant polypeptides against potential pandemic H7N9 influenza infection followed by GBS burden. Mice were intranasally immunized using 107 50% egg infectious dose (EID 50 ) of H7N3 LAIV, the mix of the 4 GBS peptides (group B streptococcus vaccine [GBSV]), or combined LAIV + GBSV vaccine. The LAIV raised serum hemagglutination-inhibition antibodies against H7N9 in higher titers than against H7N3. Combined vaccination provided advantageous protection against infections with A/Shanghai/2/2013(H7N9)CDC-RG influenza and serotype II GBS. Combined vaccine significantly improved bacterial clearance from the lungs after infection compared with other vaccine groups. The smallest lung lesions due to combined LAIV + GBSV vaccination were associated with a prevalence of lung interferon-γ messenger RNA expression. Thus, combined viral and bacterial intranasal immunization using H7N3 LAIV and recombinant bacterial polypeptides induced balanced adaptive immune response, providing protection against potential pandemic influenza H7N9 and bacterial complications., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Secondary bacterial influenza complications are a common cause of excesses morbidity and mortality, which determines the need to develop means for specific prophylaxis. Group B streptococcal infection is especially common cause of pneumonia among children and the elderly with underlying conditions. Here we investigate in a mouse model the effects of combined intranasal immunization using live attenuated influenza vaccine and recombinant polypeptides based on group B Streptococcus surface proteins., Methods: Groups of outbred mice received two doses of the following preparations: 1) the reassortant A/17/Mallard/Netherlands/00/95 (H7N3) influenza virus; 2) a mixture of P6, ScaAB, ScpB1 and Stv recombinant GBS proteins (20 µg total); 3) the A(H7N3) influenza vaccine pooled with the four bacterial peptide preparation; 4) control animals were treated with PBS., Results: Intranasal vaccination using LAIV in combination with GBS polypeptides provided advantageous protection against infections with homologous A/Mallard/Netherlands/12/00 (H7N3) wild type virus or heterologous A/Puerto Rico/8/34 (H1N1) followed by serotype II GBS infection. Also, combined vaccination improved bacterial clearance from the lungs of mice., Conclusion: Intranasal immunization with LAIV+GBSV was safe and enabled to induce the antibody response to each of vaccine components. Thus, the combined vaccine increased the protective effect against influenza and its bacterial complications in mice compared to LAIV-only.

Group B streptococcal (GBS) gene encoding the putative lipoprotein and adherence factor ScaAB was cloned and expressed in E. coli. Recombinant ScaAB protein was isolated. Signal sequence of ScaAB was found to be cleaved in the E. coli host. ScaAB recombinant protein was immunogenic in mice and antibodies against this protein were discovered in mice sera after GBS infection. The perspectives of the use of ScaAB protein in GBS vaccine are discussed.

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.

Submicroscopic aspects of the adherence of Group A streptococci to HEp-2 cells and the time sequence of their further interaction with these cells were studied. The M+ variant of streptococci, characterized by the presence of filamentous protrusions on the cell wall, displayed a high capacity for adherence, in contrast to the M- variant of the same strain, where adherence was low. The first stage of the interaction between M+ variant of Group A streptococci and HEp-2 cells was adherence of the filamentous protrusions of the bacterial cell wall to host cell cytoplasmic membrane; this was followed by closer contact of the streptococcus cell wall with HEp-2 cell surface. Continuing incubation led to the development of invaginations in the cytoplasmic membranes of HEp-2 cells, into which streptococci gradually penetrated. Ingestion of streptococci into the forming pseudovacuoles of the host cell was accompanied by bacterial cell division, culminating in total disintegration of the host cell and release of the streptococci into the medium. At all stages of the interaction there was a pronounced tendency to form multiple contacts between the surface structures of the streptococcus cell and the membrane structures of the animal cell being attacked.

Using the HEp-2 cell line system the factors and mechanisms of group A Streptococcus adherence had been studied. It was shown that high adherence was chiefly found in strains showing attributes of virulence (presence of M protein, growth in human blood, lethality for mice). The data supplied by experiments with pepsin and LTA suggest that there exist at least two mechanisms of adherence.

In vitro tests for adherence to human vaginal and pharyngeal epithelial cells were used to study the problem of tissue-specific tropism in group B streptococci (GBS). Twenty-two vaginal or pharyngeal clinical isolates of GBS (serotypes Ia, Ib, II, and III) were used. No significant differences in adherence to vaginal and pharyngeal epithelial cells were found between GBS from both sources: statistical analysis furnished no evidence for tissue-specific tropism. Serotype III vaginal GBS adhered better to vaginal and pharyngeal epithelial cells than did serotype III GBS strains isolated from the pharynx. However, pronounced differences in the level of adherence were found among strains of the same serotypes and from the same sources. Thus, the results obtained suggest that differences in adherence may rather be strain-dependent that type-dependent.

Eighty-nine fosfomycin-resistant mutants of group A and B streptococci were isolated. The mutants differed essentially from the parent strains in cell-wall characteristics, such as morphology of cocci and chains, sensitivity to detergents and hydrophobic properties. At the same time, the recipient activity of the investigated mutants was not changed. It is supposed that resistance of mutants to fosfomycin is connected with a transport block of glycerol 3-phosphate--important lipoteichoic acid (LTA) structural component. Fosr mutants can be useful for experimental examination of Beachey's hypothesis about the importance of LTA or its complex with the M protein for adhesion.

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.