Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

A broad antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that fails to protect against both seasonal antigenic variants and the next pandemic. However, antibodies directed to conserved sites can confer broad protection. We identify and characterize a class of human antibodies that engage a previously undescribed, conserved, epitope on the influenza hemagglutinin protein (HA). Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal infection in mice. Their breadth of binding extends across most influenza A serotypes and seasonal human variants. Antibodies to the head-stem interface epitope are present at low frequency in the memory B cell populations of multiple donors. The immunogenicity of the epitope warrants its consideration for inclusion in improved or "universal" influenza vaccines.

Mucosal plasma cells are required to protect the upper airway and brain from infection.

While blood antibodies mediate protective immunity in most organs, whether they protect nasal surfaces in the upper airway is unclear. Using multiple viral infection models in mice, we found that blood-borne antibodies could not defend the olfactory epithelium. Despite high serum antibody titers, pathogens infected nasal turbinates, and neurotropic microbes invaded the brain. Using passive antibody transfers and parabiosis, we identified a restrictive blood-endothelial barrier that excluded circulating antibodies from the olfactory mucosa. Plasma cell depletions demonstrated that plasma cells must reside within olfactory tissue to achieve sterilizing immunity. Antibody blockade and genetically deficient models revealed that this local immunity required CD4+ T cells and CXCR3. Many vaccine adjuvants failed to generate olfactory plasma cells, but mucosal immunizations established humoral protection of the olfactory surface. Our identification of a blood-olfactory barrier and the requirement for tissue-derived antibody has implications for vaccinology, respiratory and CNS pathogen transmission, and B cell fate decisions.

Effects of Alterations to the CX3C Motif and Secreted Form of Human Respiratory Syncytial Virus (RSV) G Protein on Immune Responses to a Parainfluenza Virus Vector Expressing the RSV G Protein.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised.IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

Mannose receptor 1 mediates cellular uptake and endosomal delivery of CpG-motif containing oligodeoxynucleotides.

Recognition of microbial components is critical for activation of TLRs, subsequent innate immune signaling, and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG-motif containing oligodeoxynucleotides (CpG ODNs) is preceded by agonist endocytosis and delivery into the endolysosomes. The events that dictate this process remain largely unknown; furthermore, it is unclear whether the receptors involved in mediating uptake of exogenous DNA are conserved for both naturally derived pathogenic DNA and synthetic ODNs. In this study, we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hyporesponsive to CpG ODN but are fully responsive to bacterial DNA, thus implying that microbial recognition is not fully recapitulated by a synthetic analog. To identify the gene responsible for the CpG ODN defect, we have performed genome-wide linkage analysis. Using N2 backcross mice, we mapped the trait with high resolution to a single locus containing Mrc1 as the gene conferring the trait. We show that mannose receptor 1 (MRC1; CD206) is involved in CpG ODN uptake and trafficking in wild-derived MOLF/Ei peritoneal macrophages. Furthermore, we show that other strains of wild-derived mice also require MRC1 for CpG-induced cytokine responses. These findings reveal novel functions for MRC1 and demonstrate that wild-derived mice are important and indispensable model for understanding naturally occurring regulators of inflammatory responses in innate immune pathways.

IRAK1BP1 inhibits inflammation by promoting nuclear translocation of NF-kappaB p50.

An orchestrated balance of pro- and antiinflammatory cytokine release is critical for an innate immune response sufficient for pathogen defense without excessive detriment to host tissues. By using an unbiased forward genetic approach, we previously reported that IL-1R-associated kinase 1 binding protein 1 (IRAK1BP1) down-modulates Toll-like receptor-mediated transcription of several proinflammatory cytokines. To gain insights into the physiological relevance of the inhibitory role of IRAK1BP1 in inflammation, we generated mutant mice lacking IRAK1BP1. Here we report that IRAK1BP1 does not inhibit signaling pathways generally but rather changes the transcriptional profile of activated cells, leading to an increase in IL-10 production and promoting LPS tolerance. This shift in cytokine transcription correlates with an increased ratio of functional NF-kappaB subunit dimers comprised of p50/p50 homodimers relative to p50/p65 heterodimers. The increase in nuclear p50/p50 was consistent with the ability of IRAK1BP1 to bind to the p50 precursor molecule and IkappaB family member p105. We conclude that IRAK1BP1 functions through its effects on NF-kappaB as a molecular switch to bias innate immune pathways toward the resolution of inflammation.