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Background Currently licensed serotype-based pneumococcal vaccines are effective in preventing invasive pneumococcal diseases, but less effective in preventing non-bacteremic pneumonia and acute otitis media (AOM). We previously reported that a trivalent pneumococcal protein recombinant vaccine (PPrV) protected against pneumonia in a murine model. Here we evaluated PPrV protection against AOM in an infant murine model. Methods C57BL/6J mice were intramuscularly vaccinated at 1–3 weeks of age with monovalent pneumococcal histidine triad protein D (PhtD), or pneumococcal choline binding protein A (PcpA), or detoxified pneumolysin (PlyD1), or trivalent vaccine, and transtympanically challenged at 7–8 weeks of age with 1 × 10 2 CFU of pneumococcal strain BG7322 (6A) or 1 × 10 4 CFU of pneumococcal nontypeable strain 0702064 MEF. Serum IgG titers were determined by ELISA. At 24 and 48 h post infection (hpi), animals were sacrificed and middle ear fluid (MEF) samples were collected to determine pneumococcal CFUs . Results We found that vaccination of infant mice with monovalent and trivalent pneumococcal proteins elicited significant serum IgG antibody responses to corresponding component proteins. Vaccination with PhtD reduced BG7322 bacterial burdens in MEF at both 24 (p = 0.05) and 48 hpi (p = 0.16). Vaccination with PcpA significantly reduced the bacterial burdens in MEF at both 24 (p = 0.02) and 48 hpi (p = 0.004), and PlyD1 significantly reduced bacterial burden in MEF at 48 hpi (p = 0.02). Vaccination with trivalent PPrV (PhtD, PcpA and PlyD1) significantly reduced Spn burdens in MEF at both 24 (p = 0.001) and 48 hpi (p Spn strain. Vaccinated mice had significantly milder inflammatory cytokine levels (IL-1β, IL-6, TNF-α, MIP-2 and KC) in middle ears at 24 hpi (all p values Conclusion Trivalent PPrV confers protection against pneumococcal AOM in an infant murine model.

Purpose. Nontypeable Haemophilus influenzae (NTHi) is a commensal in the human nasopharynx and the cause of pneumonia, meningitis, sinusitis, acute exacerbations of chronic obstructive pulmonary disease and acute otitis media (AOM). AOM is the most common ailment for which antibiotics are prescribed in the United States. With the emergence of new strains of antibiotic-resistant bacteria, finding an effective and broad coverage vaccine to protect against AOM-causing pathogens has become a priority. Mouse models are a cost-effective and efficient way to help determine vaccine efficacy. Here, we describe an NTHi AOM model in C57BL/6J mice, which also utilizes a mouse-adapted H1N1 influenza virus to mimic human coinfection. Methodology. We tested our coinfection model using a protein vaccine formulation containing protein D, a well-studied NTHi vaccine candidate that can be found in the 10-valent Streptococcus pneumoniae conjugate vaccine. We verified the usefulness of our mouse model by comparing bacterial loads in the nose and ear between protein D-vaccinated and control mice. Results. While there was no measurable difference in nasal bacterial loads, we did detect significant differences in the bacterial loads of ear washes and ear bullae between vaccinated and control mice. Conclusion. The results from this study suggest that our NTHi AOM coinfection model is useful for assessing protein vaccines.

Respiratory viral infections, such as influenza, can lead to delayed viral clearance in toddlers, possibly exacerbating disease morbidity. We hypothesized that defective CD4 T cells in toddlers may contribute to a failure to clear virus at a similar rate to adults. Thus, we developed a young mouse model to examine potential divergent responses between toddlers and adults. We determined that young mice (toddler mice, 21 d old) were actively generating and recruiting effector/memory T cells, whereas memory populations were firmly established in older, adult mice (8–10 wk old). We infected toddler and adult mice with influenza A/PR8/34 (H1N1) and found young mice had elevated morbidity, as measured by enhanced weight loss and lower partial pressure of oxygen levels, throughout the infection, thus, modeling the higher morbidity observed in children (

Respiratory syncytial virus (RSV) is a major cause of respiratory tract disease in infants, aged adults, and immunosuppressed patients. The only approved medicines for RSV disease are administration of prophylatic antibodies or treatment with a synthetic nucleoside. Both approaches are expensive and the latter is not without risk and of controversial benefit. The present investigation studied whether pharmaceutical or biologic compounds based upon chemokines might be useful in preventing RSV disease. Of interest was RANTES/CCL5, which inhibits infection by HIV strains that use chemokine receptor (CCR)-5 as co-receptor. Herein, we report that prior or simultaneous treatment of HEp-2 cells with recombinant human CCL5 provides dose-dependent inhibition of infection with RSV. Other recombinant chemokines (MIP-1α/CCL3, MIP-1β/CCL4, MCP-2/CCL8, eotaxin/CCL11, MIP-1δ/CCL15, stromal cell derived factor (SDF)-1α/CXCL12) were not inhibitory. The data suggested that CCL5 might inhibit infection by blocking fusion (F) protein–epithelial cell interactions. Infections by mutant RSV strains deleted of small hydrophobic and/or attachment proteins and only expressing F protein in the envelope were inhibited by prior treatment with CCL5 or a biologically inactive N-terminally modified met-CCL5. Inhibition was also observed when virus adsorption and treatment with CCL5 were performed at 4°C. Flow cytometry further revealed that epithelial cells were positive for CCR3, but not CCR1 or CCR5. Thus, novel mimetics of CCL5 may be useful prophylatic agents to prevent respiratory tract disease caused by RSV. J. Med. Virol. 73:300–308, 2004. © 2004 Wiley-Liss, Inc.

Immunization with native fusion (F) protein from respiratory syncytial virus (RSV) adsorbed to alum adjuvant generates greater than fourfold rises in serum neutralizing antibody titers in approximately 50% of seropositive humans. Using BALB/c mice we demonstrate herein that enhanced neutralization titers and accelerated clearance of virus from the lungs after challenge are possible if the attachment (G) glycoprotein is added to F protein-based vaccines. We further reveal for the first time that polarized type 2 T cell responses and immunopathology associated with G protein are inhibited by adjuvants recognized by toll-like receptors (TLR). Co-formulation with compounds that targeted TLR-2, TLR-3, TLR-4, or TLR-9 elicited significantly diminished type 2 T cell responses that caused granulocytic inflammation and eosinophilia in the airways after challenge. These results were not observed with recombinant IL-12 or QS-21. The data are important for improving combination vaccines for RSV.

Development of subunit vaccines against respiratory syncytial virus (RSV) for naive human infants is hindered by concerns that immunization with the fusion or attachment (G) proteins will elicit polarized Type 2 T cell responses and cause immunopotentiation upon subsequent natural infection. We investigated the regions of G protein responsible for inducing a Type 2 T cell phenotype in inbred mice of different MHC haplotype toward development of vaccines with improved safety. As demonstrated by IL-5-dependent pulmonary eosinophilia after challenge and serum anti-G protein IgG1 to IgG2 ratios, highly purified native G protein sensitized all strains for a Type 2 T cell phenotype. Stimulation of G protein-primed splenocytes with synthetic overlapping peptides indicated that the nonglycosylated ectodomain was primarily responsible. Respectively the recall responses of BALB/c (H2(d)), C57BL/6 (H-2(b)), SJL (H-2(s)), and C3H/HeJ (H-2(k)) mice were directed against epitopes within peptides spanning amino acids 184-198 (pep(184-198)), 168-181 (pep(168-181)) or 171-185 (pep(171-185)), 176-190 (pep(176-190)), and 104-118 (pep(104-118)) or 159-173 (pep(159-173)). Injection of pep(184-198) conjugated to KLH (pep(184-198)-KLH) primed H2(d) [BALB/c, B6.C-H2(d)/bBy], but not H-2(b) [C57Bl/6, C.B10-H2(b)/LiMcd] mice for pulmonary eosinophilia. Sensitization with a peptide-KLH conjugate encompassing amino acids 149-200 (pep(149-200)-KLH) further confirmed that Type 2 T cell responses in BALB/c, C57BL/6 and SJL, but not C3H/HeJ mice were induced by the nonglycosylated ectodomain of G protein. These data are important for design of safe and efficacious subunit and attenuated vaccines for RSV.

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.

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-log 10 -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.

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 cpts 248/404 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.

A history of acute bronchiolitis in infancy caused by respiratory syncytial virus is a risk factor for recurrent wheezing in early childhood. Because the attachment (G) protein sensitizes mice for pulmonary eosinophilia and because Th2 cells are central in the pathogenesis of asthma, plasma and peripheral blood mononuclear cells (PBMC) from donors with asthma and from healthy donors were evaluated for anti–G protein responses. A significant trend connecting severity of asthma with anti– G protein IgG1 and IgG2 titers was observed. The correlation between anti– F protein IgG3 titers and asthma severity approached significance. Peptide mapping studies revealed that more positive recall responses (interferon-g and interleukin-10 secretion) occurred after PBMC from donors with asthma were stimulated with peptides representing the nonglycosylated domain of G protein. The same peptides elicited more positive recall responses (proliferation and interferon-g secretion) in the PBMC of healthy donors. These data suggest that a mechanism may exist whereby adaptive immune responses against G protein contribute to wheezing. Asthma is an inflammatory disease of the airways that has a significant impact on health care costs in the United States [1]. One recognized factor contributing to asthma is viral infection of the lower respiratory tract [2]. For infants and toddlers, the virus most often associated with wheezing is respiratory syncytial virus (RSV). A recent review [3] of both prospective and retrospective studies that spanned several decades indicates that lower respiratory tract infection with RSV is a major risk factor for wheezing in children until age 11 years. RSV is an enveloped, negative-strand RNA pneumovirus estimated to cause as many as 90% of cases of bronchiolitis in infancy and 50% of all cases of pneumonia during the first 2 years of life [4].

The feasibility of using oligodeoxynucleotides (ODN) containing unmethylated CpG motifs as parenteral adjuvants for subunit vaccines against RSV was tested in BALB/c mice. Compared with immunization with natural F protein adsorbed to aluminum hydroxide (F/AlOH) adjuvant alone, coadministration of F/AlOH with CpG ODN resulted in statistically significant increases in serum neutralization titers, an enhanced generation of splenic antigen-dependent killer cell precursors, and accelerated clearance of infectious virus from lungs 4 days after challenge. The statistically significant increases in serum IFNgamma and anti-F protein IgG2a titers, and significantly diminished pulmonary IL-5 and eosinophilia after challenge indicated that CpG ODN enhanced the ability of F/AlOH to elicit type 1 immune responses. F protein-specific serum IgE titers were also reduced. Further analysis of pulmonary inflammatory cells demonstrated an expansion of CD8(+) T cells, relative to the CD4(+) T cell compartment. The potency of CpG ODN was not adversely affected in gene knockout mice devoid of the p35 chain of the IL-12 heterodimer. Taken together, the results suggest a novel formulation for naive recipients of F protein-based subunit vaccines that does not result in a type 2 phenotype.