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Background Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody’s therapeutic potential for targeting IL25 in inflammation. Results A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.

Initial promising results with immune sera guided early human mAb approaches against Gram-negative sepsis to an LPS neutralization mechanism, but these efforts failed in human clinical trials. Emergence of multidrug resistance has renewed interest in pathogen-specific mAbs. We utilized a pair of antibodies targeting Klebsiella pneumoniae LPS, one that both neutralizes LPS/TLR4 signaling and mediates opsonophagocytic killing (OPK) (54H7) and one that only promotes OPK (KPE33), to better understand the contribution of each mechanism to mAb protection in an acutely lethal pneumonia model. Passive immunization 24 hours prior to infection with KPE33 protected against lethal infection significantly better than 54H7, while delivery of either mAb 1 hour after infection resulted in similar levels of protection. These data suggest that early neutralization of LPS-induced signaling limits protection afforded by these mAbs. LPS neutralization prevented increases in the numbers of γδT cells, a major producer of the antimicrobial cytokine IL-17A, the contribution of which was confirmed using il17a-knockout mice. We conclude that targeting LPS for OPK without LPS signaling neutralization has potential to combat Gram-negative infection by engaging host immune defenses, rather than inhibiting beneficial innate immune pathways.

Receptor for advanced glycation end-products (RAGE) detects nucleic acids and promotes DNA uptake into endosomes, which in turn lowers the immune recognition threshold for TLR9 activation., Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen.

The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K. pneumoniae Several monoclonal antibodies were isolated from phage display and hybridoma platforms by functional screening for opsonophagocytic killing activity. We further identified their common target antigen to be MrkA, a major protein in the type III fimbriae complex, and showed that these serotype-independent anti-MrkA antibodies reduced biofilm formation in vitro and conferred protection in multiple murine pneumonia models. Importantly, mice immunized with purified MrkA proteins also showed reduced bacterial burden following K. pneumoniae challenge. Taken together, these results support MrkA as a promising target for K. pneumoniae antibody therapeutics and vaccines.

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the β4–β5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49GB6 and a Ly-49GB6-like receptor, Ly-49Mnonobese diabetic, but not Ly-49GBALB/c. By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the β4–β5 loop and the β1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49GB6 ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the β4–β5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd α1/α2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.

Classical class I major histocompatibility complex (MHC) molecules, as well as the nonclassical class I histocompatibility leukocyte antigen (HLA)-E molecule, can negatively regulate natural killer (NK) cell cytotoxicity through engagement of NK inhibitory receptors. We show that expression of murine (m)CD1.1, a nonpolymorphic nonclassical MHC class I–like molecule encoded outside the MHC, protects NK-sensitive RMA/S target cells from adherent lymphokine-activated killer cell (A-LAK) cytotoxicity. Passage of effector cells in recombinant interleukin (rIL)-2 enhanced protection by mCD1.1, suggesting an expansion of relevant A-LAK population(s) or modulation of A-LAK receptor expression. Murine CD1.1 conferred protection from lysis by rIL-2–activated spleen cells of recombination activating gene (Rag)-1−/− mice, which lack B and T cells, demonstrating that mCD1.1 can protect RMA/S cells from lysis by NK cells. An antibody specific for mCD1.1 partially restored A-LAK lysis of RMA/S.CD1.1 transfectants, indicating that cell surface mCD1.1 can confer protection from lysis; therefore, mCD1.1 possibly acts through interaction with an NK inhibitory receptor. CD1.1 is by far the most divergent class I molecule capable of regulating NK cell activity. Finally, mCD1.1 expression rendered RMA/S cells resistant to lysis by A-LAK of multiple mouse strains. The conserved structure of mCD1.1 and pattern of mCD1.1 resistance from A-LAK lysis suggest that mCD1.1 may be a ligand for a conserved NK inhibitory receptor.