Brady J. Hurtgen

An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection

Coccidioides is a fungal pathogen and causative agent of a human respiratory disease against which no clinical vaccine exists. In this study we evaluated a novel vaccine adjuvant referred to as EP67, which is a peptide agonist of the biologically active C-terminal region of human complement component C5a. The EP67 peptide was conjugated to live spores of an attenuated vaccine strain (ΔT) of Coccidioides posadasii. The non-conjugated ΔT vaccine provided partial protection to BALB/c mice against coccidioidomycosis. In this report we compared the protective efficacy of the ΔT-EP67 conjugate to the ΔT vaccine in BALB/c mice. Animals immunized subcutaneously with the ΔT-EP67 vaccine showed significant increase in survival and decrease in fungal burden over 75 days postchallenge. Increased pulmonary infiltration of dendritic cells and macrophages was observed on day 7 postchallenge but marked decrease in neutrophil numbers had occurred by 11 days. The reduced influx of neutrophils may have contributed to the observed reduction of inflammatory pathology. Mice immunized with the ΔT-EP67 vaccine also revealed enhanced expression of MHC II molecules on the surface of antigen presenting cells, and in vitro recall assays of immune splenocytes showed elevated Th1- and Th17-type cytokine production. The latter correlated with a marked increase in lung infiltration of IFN-γ- and IL-17-producing CD4(+) T cells. Elevated expression of T-bet and RORc transcription factors in ΔT-EP67-vaccinated mice indicated the promotion of Th1 and Th17 cell differentiation. Higher titers of Coccidioides antigen-specific IgG1 and IgG2a were detected in mice immunized with the EP67-conjugated versus the non-conjugated vaccine. These combined results suggest that the EP67 adjuvant enhances protective efficacy of the live vaccine by augmentation of T-cell immunity, especially through Th1- and Th17-mediated responses to Coccidioides infection.

Volumetric muscle loss: persistent functional deficits beyond frank loss of tissue.

Open fracture is a common occurrence in civilian and military populations. Though great strides have been made in limb salvage efforts, persistent muscle strength deficits can contribute to a diminished limb function after the bone has healed. Over the past decade, a growing effort to establish therapies directed at de novo muscle regeneration has produced several therapeutic approaches. As this effort progresses and as therapies reach clinical testing, many questions remain regarding the pathophysiology of the volumetric loss of skeletal muscle. The current study demonstrates, in a rat “open fracture” model, that the volumetric loss of skeletal muscle results in persistent functional deficits that are dependent on muscle length and joint angle. Moreover, the injured muscle has an increased stiffness during passive stretch and a reduced functional excursion. A case study of a patient with an open type III tibia fracture resulting in volumetric muscle loss in the anterior and posterior compartment is also presented. Eighteen months after injury and tibia healing, persistent functional deficits are apparent with many of the same qualities demonstrated in the animal model. Muscle architectural adaptations likely underlie the altered intrinsic functional characteristics of the remaining musculature. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 33:40–46, 2015.

Construction and Evaluation of a Novel Recombinant T Cell Epitope-Based Vaccine against Coccidioidomycosis

ABSTRACT Clinical and animal studies of coccidioidomycosis have demonstrated that activated CD4+ T lymphocytes are essential for protection against this fungal respiratory disease. We previously reported a vaccine against Coccidioides infection which contained three recombinant CD4+ T cell-reactive proteins and induced a robust, protective immune response in mice. Due to the anticipated high cost of production and clinical assessment of this multivalent vaccine, we generated a single protein which contained immunodominant T cell epitopes of the three polypeptides. Epitopes were initially identified by computational prediction of their ability to bind promiscuously to human major histocompatibility complex class II (MHC II) molecules. Cellular immunoassays confirmed the immunogenicity of the synthesized epitope peptides, while in vitro binding assays revealed a range of peptide affinity for MHC II. A DNA construct was synthesized for bacterial expression of a recombinant protein vaccine which contained five epitopes with the highest affinity for human MHC II, each fused with leader and spacer peptides proposed to optimize epitope processing and presentation to T cell receptors. Recall assays of immune T lymphocytes obtained from human MHC II-expressing HLA-DR4 transgenic mice confirmed that 4 of the 5 epitope peptides were processed. Mice immunized with the epitope-based vaccine admixed with a synthetic oligodeoxynucleotide adjuvant or loaded into yeast glucan particles and then challenged intranasally with Coccidioides showed early lung infiltration of activated T helper-1 (Th1), Th2, and Th17 cells, elevated gamma interferon (IFN-γ) and interleukin (IL)-17 production, significant reduction of fungal burden, and prolongation of survival compared to nonvaccinated mice. This is the first report of an epitope-based vaccine against coccidioidomycosis.

Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro

BackgroundOsteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).MethodsOsteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.ResultsBCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.ConclusionsTogether these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Biofilm and planktonic pneumococci demonstrate disparate immunoreactivity to human convalescent sera

BackgroundStreptococcus pneumoniae (the pneumococcus) is the leading cause of otitis media, community-acquired pneumonia (CAP), sepsis, and meningitis. It is now evident that S. pneumoniae forms biofilms during nasopharyngeal colonization; the former which facilitates persistence, the latter, a prerequisite for subsequent development of invasive disease. Proteomic evaluation of S. pneumoniae suggests the antigen profile available for host-recognition is altered as a consequence of biofilm growth. This has potentially meaningful implications in regards to adaptive immunity and protection from disseminated disease. We therefore examined the antigen profile of biofilm and planktonic pneumococcal cell lysates, tested their reactivity with human convalescent sera and that generated against biofilm pneumococci, and examined whether immunization with biofilm pneumococci protected mice against infectious challenge.ResultsBiofilm pneumococci have dramatically altered protein profiles versus their planktonic counterparts. During invasive disease the humoral immune response is skewed towards the planktonic protein profile. Immunization with biofilm bacteria does not elicit a strong-cross-reactive humoral response against planktonic bacteria nor confer resistance against challenge with a virulent isolate from another serotype. We identified numerous proteins, including Pneumococcal serine-rich repeat protein (PsrP), which may serve as a protective antigens against both colonization and invasive disease.ConclusionDifferential protein production by planktonic and biofilm pneumococci provides a potential explanation for why individuals remain susceptible to invasive disease despite previous colonization events. These findings also strongly suggest that differential protein production during colonization and disease be considered during the selection of antigens for any future protein vaccine.

Novel Strategies to Enhance Vaccine Immunity against Coccidioidomycosis

Abstract : Coccidioidomycosis is a potentially life-threatening respiratory mycosis endemic to the Americas and caused by inhalation of spores produced by the molds Coccidioides immitis and C. posadasii. The dry, air-dispersed infectious spores (arthroconidia) are derived from saprobic-phase filaments that grow in semidesert soil of the southwestern United States and arid regions of Mexico and Central and South America [1]. Coccidioides is a dimorphic ascomycetous fungus with distinct saprobic and parasitic phases and is classified in the order Onygenales together with other genera of pathogenic molds that include Histoplasma, Blastomyces, and Paracoccidioides. The clinical spectrum of disease caused by these environmental pathogens ranges from a mild infection that resolves spontaneously to a disseminated mycosis. Inhaled spores of Coccidioides are small enough to colonize the lowermost reaches of the respiratory tree. Nonhuman primates experimentally challenged by aerosolization with fewer than ten infectious propagules developed a severe form of the disease and died within four to six weeks [2]. The parasitic cycle of Coccidioides is unique among these medically important, dimorphic molds. Spores in the lungs germinate to form multinucleate spherules (.60 mm diameter) that are too large to be engulfed by host phagocytes. The contents of mature spherules differentiate into a multitude of propagative endospores. Production of indigestible spherules that display high fecundity during the endosporulation stage confound phagocytic defenses and augment survival of the fungus in the lungs. Mammalian innate immune defenses are further compromised by the ability of endosporulating spherules to generate an alkaline microenvironment at infection sites due to their release of ammonia, which contributes to the pathogen s virulence [3].

Progress Toward a Human Vaccine Against Coccidioidomycosis

Coccidioidomycosis (San Joaquin Valley fever) is a human respiratory disease caused by a soil-borne mold, and is recognized as an intransigent microbial infection by physicians who treat patients with the potentially life-threatening, disseminated form of this mycosis. Epidemiological studies based on surveys of skin-test reactivity of people who reside in the endemic regions of the Southwestern US have shown that at least 150,000 new infections occur annually. The clinical spectrum of coccidioidomycosis ranges from an asymptomatic insult to a severe pulmonary disease in which the pathogen may spread from the lungs to the skin, bones, brain and other body organs. Escalation of symptomatic infections and increased cost of long-term antifungal treatment warrant a concerted effort to develop a vaccine against coccidioidomycosis. This review examines recently reported strategies used to generate such a vaccine and summarizes current understanding of the nature of protective immunity to this formidable disease.

Autologous minced muscle grafts improve endogenous fracture healing and muscle strength after musculoskeletal trauma

The deleterious impact of concomitant muscle injury on fracture healing and limb function is commonly considered part of the natural sequela of orthopedic trauma. Recent reports suggest that heightened inflammation in the surrounding traumatized musculature is a primary determinant of fracture healing. Relatedly, there are emerging potential therapeutic approaches for severe muscle trauma (e.g., volumetric muscle loss [VML] injury), such as autologous minced muscle grafts (1 mm3 pieces of muscle; GRAFT), that can partially prevent chronic functional deficits and appear to have an immunomodulatory effect within VML injured muscle. The primary goal of this study was to determine if repair of VML injury with GRAFT rescues impaired fracture healing and improves the strength of the traumatized muscle in a male Lewis rat model of tibia open fracture. The most salient findings of the study were: (1) tibialis anterior (TA) muscle repair with GRAFT improved endogenous healing of fractured tibia and improved the functional outcome of muscle regeneration; (2) GRAFT repair attenuated the monocyte/macrophage (CD45+CDllb+) and T lymphocyte (CD3+) response to VML injury; (3) TA muscle protein concentrations of MCP1, IL‐10, and IGF‐1 were augmented in a proregenerative manner by GRAFT repair; (4) VML injury concomitant with osteotomy induced a heightened systemic presence of alarmins (e.g., soluble RAGE) and leukocytes (e.g., monocytes), and depressed IGF‐1 concentration, which GRAFT repair ameliorated. Collectively, these data indicate that repair of VML injury with a regenerative therapy can modulate the inflammatory and regenerative phenotype of the treated muscle and in association improve musculoskeletal healing.

Preclinical identification of vaccine induced protective correlates in human leukocyte antigen expressing transgenic mice infected with Coccidioides posadasii.

There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB1∗0401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4+ T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50-70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9dpc. While contraction rates of Th cells and the inflammatory response during 14-35dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9-14days. The FAD group approached moribund by 14dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.

Rational Design of T Lymphocyte Epitope-Based Vaccines Against Coccidioides Infection

Coccidioidomycosis is a potentially life-threatening mycosis endemic to the Southwestern USA and some arid regions of Central and South America. A vaccine against Coccidioides infection would benefit over 30-million people who reside in or visit the endemic regions. Vaccine candidates against systemic fungal infections come in many forms. Live attenuated vaccines are derived from disease-causing pathogens and generally stimulate excellent protective immunity. Since attenuated vaccines contain living microbes, there is a degree of unpredictability raising concerns regarding safety and stability. Generation of a subunit vaccine has initiated efforts to design a safe reagent suitable for administration to humans at risk of coccidioidomycosis. Epitope-based vaccines allow for eliciting specific protective immune responses and removal of potentially detrimental sequences to improve safety. This chapter describes methods for the identification of T cell epitopes derived from Coccidioides antigens, design, and production of a recombinant vaccine containing multiple T cell epitopes, and evaluation of its protective efficacy and vaccine immunity against pulmonary Coccidioides infection using a strain of transgenic mice that express a human MHC II molecule.