Search

Nontypeable Haemophilus influenzae (NTHi) has emerged as a dominant mucosal pathogen causing acute otitis media (AOM) in children, acute sinusitis in children and adults, and acute exacerbations of chronic bronchitis in adults. Consequently, there is an urgent need to develop a vaccine to protect against NTHi infection. A multi‐component vaccine will be desirable to avoid emergence of strains expressing modified proteins allowing vaccine escape. Protein D (PD), outer membrane protein (OMP) 26, and Protein 6 (P6) are leading protein vaccine candidates against NTHi. In pre‐clinical research using mouse models, we found that recombinantly expressed PD, OMP26, and P6 induce robust antibody responses after vaccination as individual vaccines, but when PD and OMP26 were combined into a single vaccine formulation, PD antibody levels were significantly lower. We postulated that PD and OMP26 physiochemically interacted to mask PD antigenic epitopes resulting in the observed effect on antibody response. However, column chromatography and mass spectrometry analysis did not support our hypothesis. We postulated that the effect might be in vivo through the mechanism of protein vaccine immunologic antigenic competition. We found when PD and OMP26 were injected into the same leg or separate legs of mice, so that antigens were immunologically processed at the same or different regional lymph nodes, respectively, antibody levels to PD were significantly lower with same leg vaccination. Different leg vaccination produced PD antibody levels quantitatively similar to vaccination with PD alone. We conclude that mixing PD and OMP26 into a single vaccine formulation requires further formulation studies.

Nontypeable Haemophilus influenzae (NTHi) has emerged as a dominant mucosal pathogen causing acute otitis media (AOM) in children, acute sinusitis in children and adults, and acute exacerbations of chronic bronchitis in adults. Consequently, there is an urgent need to develop a vaccine to protect against NTHi infection. A multi‐component vaccine will be desirable to avoid emergence of strains expressing modified proteins allowing vaccine escape. Protein D (PD), outer membrane protein (OMP) 26, and Protein 6 (P6) are leading protein vaccine candidates against NTHi. In pre‐clinical research using mouse models, we found that recombinantly expressed PD, OMP26, and P6 induce robust antibody responses after vaccination as individual vaccines, but when PD and OMP26 were combined into a single vaccine formulation, PD antibody levels were significantly lower. We postulated that PD and OMP26 physiochemically interacted to mask PD antigenic epitopes resulting in the observed effect on antibody response. However, column chromatography and mass spectrometry analysis did not support our hypothesis. We postulated that the effect might be in vivo through the mechanism of protein vaccine immunologic antigenic competition. We found when PD and OMP26 were injected into the same leg or separate legs of mice, so that antigens were immunologically processed at the same or different regional lymph nodes, respectively, antibody levels to PD were significantly lower with same leg vaccination. Different leg vaccination produced PD antibody levels quantitatively similar to vaccination with PD alone. We conclude that mixing PD and OMP26 into a single vaccine formulation requires further formulation studies. [ABSTRACT FROM AUTHOR]

Abstract Background In the current transmission, we studied the immunogenicity and protective efficacy of serotypes 22F and 33F in the prevention of colonization and of invasive Streptococcus pneumoniae ( Spn ) pathogenesis during an influenza co-infection. Serotypes 22F and 33F are emerging Spn serotypes, which are not part of currently administered pneumococcal conjugate vaccine formulations (PCVs). Spn serotype 6A is an ingredient in the currently administered PCV13 vaccine and was therefore included in the study as a control. Methods Adult (six weeks) and infant (two weeks) C57BL/6 mice were intranasally infected in the nasopharynx (NP) with Spn serotypes 22F, 33F, or 6A. Influenza A H1N1 A/Puerto Rico/8/193 virus (PR8) was introduced one day after the NP Spn colonization. In an immunization challenge study, mice were vaccinated with monovalent 22F, 33F, or 6A polysaccharide conjugated to the CRM 197 antigen. The immunized mice were colonized or co-infected to study the vaccines efficacy. Results All three Spn serotypes established colonization in adult and infant mice. The co-infected mice showed an increase in Spn NP density. Invasive Spn infection (bacteremia) was observed following the co-infection with serotypes 22F and 6A but not 33F in adult mice, whereas infant mice developed bacteremia following co-infection with all three Spn serotypes. The vaccinations led to robust serum antibody responses to capsular polysaccharides 22F, 6A, and less for 33F. The vaccinations resulted in reductions of Spn NP colonization density for all three serotypes, prevention of bacteremia, and increased survival with Spn serotypes 22F and 6A. Passive transfer of antisera was associated with a reduction of Spn colonization densities in infant mice. Conclusion Vaccinations with monovalent 22F, 33F, or 6A formulations protect against Spn colonization, and the efficacy of the 22F vaccination was comparable to the 6A vaccination in preventing an invasive Spn bacterial infection during an influenza co-infection. [ABSTRACT FROM AUTHOR]

Respiratory syncytial (RSV) and parainfluenza (PIV) viruses are primary causes of acute bronchiolitis and wheezing illnesses in infants and young children. To further understand inflammation in the airways following infection, we tested for the presence of matrix metalloproteinases (MMP) and natural tissue inhibitors of MMP (TIMP) in primary and established human cell lines, and in the nasopharyngeal secretions (NPS) of human infants infected with RSV or PIV. Using ELISA and multiplex‐based assays, MMP‐9 and TIMP‐1 proteins were, respectively, detected in 66/67 and 67/67 NPS. During PIV or RSV infection TIMP‐1 concentrations were associated with hypoxic bronchiolitis. TIMP‐1 amounts were also negatively correlated with O2 saturation, and positively correlated with IL‐6, MIP‐1α, and G‐CSF amounts following RSV infection. IL‐6, MIP‐1α, and G‐CSF were negatively correlated with O2 saturation during RSV infection. Acute respiratory tract disease was not associated with MMP‐9 protein/protease activity. Additional studies using real‐time quantitative PCR suggested that MMP‐9 mRNA copy numbers were elevated in normal human bronchial epithelial (NHBE) cells infected with RSV, while TIMP‐1 and TIMP‐2 were not increased. However, ELISA did not reveal MMP‐9 protein in the NHBE cell culture supernatants. Hence, the data implied that airway epithelial cells were not the primary source of MMP or TIMP following paramyxovirus infection. Taken together, the data suggested that paramyxovirus infection perturbs MMP‐9/TIMP‐1 homeostasis that in turn may contribute to the severity of respiratory tract disease. J. Med. Virol. 79:447–456, 2007. © 2007 Wiley‐Liss, Inc. [ABSTRACT FROM AUTHOR]

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpΔG150-222 or rA2cpΔG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37°C, but pinpoint morphology was observed at 39°C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log10-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpΔG150-222 or rA2cpΔG177-220. These findings are important for the development of attenuated RSV vaccines. [ABSTRACT FROM AUTHOR]

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpΔG118, rA2cpΔG174, rA2cpΔG193, and rA2cpΔG213. All rA2cpΔG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37°C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpΔG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpΔG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/ 404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpΔG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV. [ABSTRACT FROM AUTHOR]