Influenza-induced immune suppression to methicillin-resistant Staphylococcus aureus is mediated by TLR9.

Bacterial lung infections, particularly with methicillin-resistant Staphylococcus aureus (MRSA), increase mortality following influenza infection, but the mechanisms remain unclear. Here we show that expression of TLR9, a microbial DNA sensor, is increased in murine lung macrophages, dendritic cells, CD8+ T cells and epithelial cells post-influenza infection. TLR9-/- mice did not show differences in handling influenza nor MRSA infection alone. However, TLR9-/- mice have improved survival and bacterial clearance in the lung post-influenza and MRSA dual infection, with no difference in viral load during dual infection. We demonstrate that TLR9 is upregulated on macrophages even when they are not themselves infected, suggesting that TLR9 upregulation is related to soluble mediators. We rule out a role for elevations in interferon-γ (IFNγ) in mediating the beneficial MRSA clearance in TLR9-/- mice. While macrophages from WT and TLR9-/- mice show similar phagocytosis and bacterial killing to MRSA alone, following influenza infection, there is a marked upregulation of scavenger receptor A and MRSA phagocytosis as well as inducible nitric oxide synthase (Inos) and improved bacterial killing that is specific to TLR9-deficient cells. Bone marrow transplant chimera experiments and in vitro experiments using TLR9 antagonists suggest TLR9 expression on non-hematopoietic cells, rather than the macrophages themselves, is important for regulating myeloid cell function. Interestingly, improved bacterial clearance post-dual infection was restricted to MRSA, as there was no difference in the clearance of Streptococcus pneumoniae. Taken together these data show a surprising inhibitory role for TLR9 signaling in mediating clearance of MRSA that manifests following influenza infection.

Cutting Edge: Check Your Mice-A Point Mutation in the Ncr1 Locus Identified in CD45.1 Congenic Mice with Consequences in Mouse Susceptibility to Infection.

B6.SJL-Ptprca Pepcb /Boy (CD45.1) mice have been used in hundreds of congenic competitive transplants, with the presumption that they differ from C57BL/6 mice only at the CD45 locus. In this study, we describe a point mutation in the natural cytotoxicity receptor 1 (Ncr1) locus fortuitously identified in the CD45.1 strain. This point mutation was mapped at the 40th nucleotide of the Ncr1 locus causing a single amino acid mutation from cysteine to arginine at position 14 from the start codon, resulting in loss of NCR1 expression. We found that these mice were more resistant to CMV due to a hyper innate IFN-γ response in the absence of NCR1. In contrast, loss of NCR1 increased susceptibility to influenza virus, a result that is consistent with the role of NCR1 in the recognition of influenza Ag, hemagglutinin. This work sheds light on potential confounding experimental interpretation when this congenic strain is used as a tool for tracking lymphocyte development.

Integrin alphavbeta1 promotes infection by human metapneumovirus.

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for alphavbeta1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with alphav or beta1 cDNAs confers hMPV infectivity, whereas reduction of alphav and beta1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that alphavbeta1 integrin is a functional receptor for hMPV.

An alphavirus replicon-based human metapneumovirus vaccine is immunogenic and protective in mice and cotton rats.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that causes upper and lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. Here, we developed Venezuelan equine encephalitis virus replicon particles (VRPs) encoding hMPV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and protective efficacy of these vaccine candidates in mice and cotton rats. VRPs encoding hMPV F protein, when administered intranasally, induced F-specific virus-neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. Challenge virus replication was reduced significantly in both the upper and lower respiratory tracts following intranasal hMPV challenge in these animals. However, vaccination with hMPV G protein VRPs did not induce neutralizing antibodies or protect animals from hMPV challenge. Close examination of the histopathology of the lungs of VRP-MPV F-vaccinated animals following hMPV challenge revealed no enhancement of inflammation or mucus production. Aberrant cytokine gene expression was not detected in these animals. Together, these results represent an important first step toward the use of VRPs encoding hMPV F proteins as a prophylactic vaccine for hMPV.

A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.