Preclinical assessment of safety of maternal vaccination against respiratory syncytial virus (RSV) in cotton rats.

Maternal immunization directed to control RSV infection in newborns and infants is an appealing vaccination strategy currently under development. In this work we have modeled maternal vaccination against RSV in cotton rats (CR) to answer two fundamental questions on maternal vaccine safety. We tested (i), whether a known, unsafe RSV vaccine (i.e., FI-RSV Lot 100 vaccine) induces vaccine enhanced disease in the presence of passively transferred, RSV maternal immunity, and (ii) whether the same FI-RSV vaccine could induce vaccine enhanced disease in CR litters when used to immunize their RSV-primed mothers. Our data show that FI-RSV immunization of pups with subsequent RSV infection results in vaccine-enhanced disease independent of whether the pups were born to RSV-seropositive or RSV-seronegative mothers, and that FI-RSV immunization of RSV-seropositive mothers does not present a health risk to either the mother or the infant. Our study also raises a novel concern regarding infant immunization, namely that "safe" RSV vaccines (e.g., live RSV administered intramuscularly) may induce vaccine-enhanced disease in RSV-infected pups born to seropositive mothers. Finally, we describe for the first time a sharp decrease in RSV neutralizing antibody titers in immunized seropositive CR at the time of delivery. This decline may reflect maternal immune suppression, potentially pinpointing a window of increased vulnerability to RSV infection that could be alleviated by effective immunization of expectant mothers.

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain.

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.

Agents that increase AAM differentiation blunt RSV-mediated lung pathology.

RSV is the most significant cause of serious lower respiratory tract infection in infants and young children worldwide. There is currently no vaccine for the virus, and antiviral therapy (e.g., ribavirin) has shown no efficacy against the disease. We reported that alternatively activated macrophages (AAMs) mediate resolution of RSV-induced pathology. AAM differentiation requires macrophage-derived IL-4 and -13, autocrine/paracrine signaling through the type I IL-4 receptor, and STAT6 activation. Based on these findings, we reasoned that it would be possible to intervene therapeutically in RSV disease by increasing AAM differentiation, thereby decreasing lung pathology. Mice treated with the IL-4/anti-IL-4 immune complexes, shown previously to sustain levels of circulating IL-4, increased the RSV-induced AAM markers arginase-1 and mannose receptor and decreased the lung pathology. Induction of PPARγ, shown to play a role in AAM development, by the PPARγ agonist rosiglitazone or treatment of mice with the macrolide antibiotic AZM, also reported to skew macrophage differentiation to an AAM phenotype, increased the AAM markers and mitigated RSV-induced lung pathology. Collectively, our data suggest that therapeutic manipulation of macrophage differentiation to enhance the AAM phenotype is a viable approach for ameliorating RSV-induced disease.

Induction of early inflammatory gene expression in a murine model of nonresuscitated, fixed-volume hemorrhage.

The etiology of many end-organ problems associated with hemorrhage has been attributed to the inflammatory response to hemorrhage. In a murine model of nonresuscitated, fixed-volume hemorrhage, we sought to elucidate the role that hemorrhagic insult alone plays in the generation of the early inflammatory cascade. Differences could be appreciated as early as 1 h post-hemorrhage, with consistent differences detected by 3 h in all of the major cytokine genes studied. Significant upregulation of IL-1beta , IL-6, TNF-alpha, and IL-10 mRNA expression was observed in both the liver and lung samples of mice subjected to fixed-volume hemorrhage when compared with sham-hemorrhaged mice. The cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) genes also were upregulated in the livers and lungs of hemorrhaged mice. Finally, expression of the genes that encode the Toll-like receptors (TLR)-2 and -4 was increased by hemorrhage. Taken collectively, these data demonstrate that the initial inflammatory cascade associated with hemorrhage occurs within hours after the initial hemorrhagic event, and can be associated with significant modulation of expression of key pro- and anti-inflammatory cytokine, enzyme, and TLR genes, suggesting that these may be possible new therapeutic targets.