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Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with a mutation known to inhibit NADase activity. We are now using TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing RT-PCR to analyze the effects of these mutant TIRs on innate cell activation. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses. Supported by grant from NIH (R25 GM119970 04)

The need for effective infectious disease vaccines has become an inescapable topic over the past year. Continued development of next-generation vaccines that provide robust protective immunity is imperative. Such vaccines will likely include an adjuvant that avoids excessive adverse reactions and allows for dose and antigen sparing. Bacterial lipid A mimetics BECC438 and BECC470 have recently emerged as lead adjuvant candidates across several experimental models of infectious disease including Yersinia pestis (plague), human papilloma virus (HPV), and influenza-A (flu). To further define BECC438 and BECC470 as immuno-adjuvants, even without antigen from an infectious pathogen, studies presented here use ovalbumin (Ova) as a model antigen in a murine prime-boost vaccine model. Higher magnitude and more balanced production of antibody isotypes IgG1 and IgG2 are observed when BECC adjuvants are compared to classic adjuvants alum and PHAD. This optimal antibody response is durable and maintained for at least 12 weeks post-vaccination. Initial experiments use C57BL6 mice and are expanded to include BALBc and CD-1 (outbred) mice. Observed immune metrics maintained similar trends across male and females, and genetic backgrounds tested. Biacore immunogenicity analysis of C57BL6 serum found an increased half-life of Ova-specific antibodies in BECC438-adjuvanted animals potentially indicative of a higher antigen binding affinity. Toxicity studies conducted in New Zealand White rabbits report that BECC438 was well tolerated with no significant reactogenicity after 50μg and 100μg intra-muscular adjuvant injection. These studies provide continued evidence supporting development of BECC adjuvants in vaccines for human use.

Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with mutations already known to inhibit NADase activity. We are now using mutant TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing reporter assays to analyze the effects of these mutant TIRs on NF-kB signaling. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity, and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.

Infectious diseases continue to be the leading cause of morbidity and mortality worldwide, an urgent need exists to develop effective vaccines against a variety of pathogens. Effective vaccines contain target pathogen antigens and adjuvant(s) which effect the magnitude, duration and mechanism of action of the immune response. Bacterial enzyme combinatorial chemistry (BECC) was developed to synthesize custom bacterial lipid A that are partial agonists for innate immune receptor Toll-like receptor 4 (TLR4). BECC is a robust mechanism for design of lipid A mimetics that drive specific activating immunity while avoiding excessive adverse host reactions. Initial screenings identify two lead adjuvant candidates, BECC438 and BECC470, that are formulated with bacterial and viral antigens, including Yersinia pestis (rF1-V), Staphlococcus alpha toxin, and HPV (VLPs). Mice are immunized on two-week intervals with several squalene or aqueous based BECC formulations. Antigen-specific serum antibody titers measured by ELISA for total IgG, IgG1, and IgG2c(2a) for BECC formulations are significantly more balanced than those currently approved for clinical use. Pathogen challenge models also show increased levels of efficacy/survival. Both BECC compounds allow for durable antibody production, antigen sparing, dose sparing, adjuvant sparing, production of T follicular helper cells, and provide protection against a heterologous challenge in viral infection models. Additionally, BECC adjuvanted vaccines with the model antigen ovalbumin show balanced and durable antibody responses in multiple murine backgrounds C57BL6, BALBc, and CD-1. BECC molecules are effective components of next-generation infectious disease vaccines.

UV radiation (UVR) induces DNA damage, leading to the accumulation of mutations in epidermal keratinocytes and immunosuppression, which contribute to the development of nonmelanoma skin cancer. We reported previously that the TLR4–MyD88 signaling axis is necessary for UV-induced apoptosis. In the dinitrofluorobenzene contact hypersensitivity model, UV-irradiated MyD88-deficient (MyD88−/−) C57BL/6 mice had intact ear swelling, exaggerated inflammation, and higher levels of dinitrofluorobenzene-specific IgG2a compared with wild-type (WT) mice. Even with normal UV-induced, dendritic cell migration, DNA damage in the local lymph nodes was less pronounced in MyD88−/− mice compared with WT mice. Cultured, UV-irradiated WT APCs showed cleavage (inactivation) of the DNA damage–recognition molecule PARP, whereas PARP persisted in MyD88−/− and TLR4−/− APCs. Epidermal DNA from in vivo UV-irradiated MyD88−/− mice had an increased resolution rate of cyclobutane pyrimidine dimers. Both in vitro treatment of MyD88−/− APCs with and intradermal in vivo injections of PARP inhibitor, PJ-34, caused WT-level cyclobutane pyrimidine dimer repair. Lymphoblasts deficient in DNA repair (derived from a xeroderma pigmentosum group A patient) failed to augment DNA repair after MyD88 knockdown after UVR, in contrast to lymphoblasts from a healthy control. These data suggest that interference with the TLR4/MyD88 pathway may be a useful tool in promoting DNA repair and maintaining immune responses following UVR-induced damage.

Dendritic cell (DC)-based immunotherapy is a promising treatment alternative for drug-resistant malignancies. Identifying candidate immunostimulatory compounds that can reliably facilitate DC maturation in the context of antigen-specific therapies is a significant hurdle in the development of such immunotherapies. In this study, we present a high throughput in vitro primary cell assay to evaluate dose-dependent DC maturation in a panel of healthy donor primary cells. CD14+ monocytes from four healthy donors were isolated from leukapheresis (Leuko Pak)-derived peripheral blood mononuclear cells (PBMC), differentiated by culture with GM-CSF and IL-4 cytokines, and then stimulated with lipopolysaccharide (LPS), CpG oligonucleotides, or a panel of novel Toll-like receptor 4 (TLR4) ligands. To quantify the resultant maturation, flow cytometry was used to measure the expression of cell surface biomarkers MHC-II, CD40, CD80, CD83, and CD86. While monocytes from all donors responded to the ligands tested, the intensity of activation marker expression varied between donors. LPS-induction of CD83 expression ranged from 27 to 82% of DCs amongst the 4 donors tested, highlighting the importance of multi-donor screening in interpreting quantitative results. In conclusion, a high throughput in vitro primary monocyte-derived DC differentiation assay reveals donor to donor variations that may go unobserved in screening experiments completed in cell lines or single donor primary cells sources. This assay can be used to assist researchers in lead generation, lead optimization, and mechanism of action studies of immunostimulatory compounds.

Adjuvant properties of bacterial cell wall components like MPLA are well described, and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential source bacterial strains, useable compounds within this promising class of adjuvants remain unfortunately low. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate functionally diverse and rationally designed lipid A. BECC removes endogenous, or introduces exogenous, lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis (Yp), to develop structurally distinct LOS molecules that elicit differential TLR4 activation. Using reporter cell lines that measure innate immune activated NF-κB transcription, BECC-derived molecules were screened for the ability to induce a lower pro-inflammatory response than E. coli LOS. Structures exhibit varied, dose-dependent TLR4-driven NF-κB activation with both human- and mouse-TLR4 complexes. Additional cytokine secretion screening identified molecules that induce comparable levels of TNF-α and IL-8, as compared to PHAD. Lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary PBMCs, and human monocyte-derived DC. This newly described BECC system allows for directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates comparable if not preferable to PHAD and MPLA.

Ultraviolet irradiation (UV) induced cellular damage is classically associated with apoptosis and known to result in systemic immunosuppression. How the decision to undergo apoptosis is made following UV is not fully understood. We hypothesize that a central mediator of TLR signaling, MyD88, determines cell fate after UV. Survival after UV of immortalized bone marrow-derived macrophages (BMM) from MyD88 germline-deficient mice (MyD88-/-) was significantly higher and apoptotic morphology reduced, vs. wild-type (WT) BMM. UV-induced DNA laddering in peritoneal macrophages (PM) and epidermis of MyD88-/- animals, vs. WT, was decreased. In MyD88-/- PM, decreased cleavage of pro-necroptotic protein RIP1, elevated mRNA levels of RIP1 and RIP3, and significantly increased TNF-α release suggests that necroptosis rather than apoptosis has been initiated. In vivo studies after UV showed significantly less TUNEL-positive cells and more profound inflammation in MyD88-/- skin sections. MyD88-/- mice are also found to be resistant to UV-induced immunosuppression. Considering that MyD88 participates in many TLR pathways, TLR2-/-, TLR4-/-, and WT BMM were compared for evidence of UV-induced apoptosis. Only TLR4-/- BMM had a similar phenotype to MyD88-/-, suggesting that the TLR4-MyD88 axis importantly contributes to cell fate decision. Our study describes a new cellular consequence of MyD88 signaling after UV, and may provide rationale for therapies to mitigate UV-induced immunosuppression.

The toll-like receptor (TLR) pathway is stimulated by pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Activation of this pathway leads to an inflammatory and immunomodulating response appropriate for the stimulus by which it was activated. Recently a body of literature has emerged indicating that TLR stimulation may not only result in an inflammatory response, but may also lead to an up-regulation of DNA repair genes and an increase in functional DNA repair after injury. These conclusions are drawn from studies conducted in the mouse animal model, however the human DNA repair response to TLR activation has yet to be investigated. Using an in vitro cell culture system, we find that human DNA repair genes are similarly up-regulated. Previously, only TLR 7 and TLR 9 agonists have been shown in mice to stimulate increased DNA repair; in this study we expand the panel of TLR agonists investigated and show that the induction of this phenomenon may not be unique to the previously studied TLR agonists. This functional increase in repair after injury may lead to a wide range of outcomes for the cells involved and warrants further study into the ever broadening role of TLR signaling in controlling cell fate.