Heterologous peptide display on chromatin nanofibers: A new strategy for peptide vaccines.

Chromatin organization starts from a "beads-on-a string" 10 nm fiber, a basic nucleosomal structure consisting of DNA and core histones. Given its regular nucleosome array on DNA backbone where N-terminal tails of each histone are exposed on the surface of chromatin fiber, we hypothesized that chromatin can be utilized as a heterologous peptide carrier to elicit a peptide-specific immune response. The plasmid DNA containing the Widom's clone 601 sequence and the recombinant chimeric histones containing the peptide derived from ras oncogene (G12V) were used to assemble the chromatin fiber in vitro. The immunogenicity of the assembled chromatin was tested in mice as a single vaccine component or formulated with adjuvants. G12V tagged-chromatin co-administered with adjuvants induced higher antibody responses against the G12V peptide than vaccination with adjuvant alone, while chimeric histones did not generate a significant antibody response. Interestingly, splenocytes from mice vaccinated with the G12V tagged-chromatin vaccine did not generate significant antigen-specific cytokine responses. Our studies suggest that chromatin can be utilized as an effective carrier of antigenic peptides for inducing specific antibody responses.

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine.

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, ß-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli-Mycobacterium shuttle plasmids and expressed in trans Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. MBB, A: E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens.IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens.

Immunogencity of antigens from Mycobacterium tuberculosis self-assembled as particulate vaccines.

Traditional approaches to vaccine development have failed to identify better vaccines to replace or supplement BCG for the control of tuberculosis (TB). Subunit vaccines offer a safer and more reproducible alternative for the prevention of diseases. In this study, the immunogenicity of bacterially derived polyester beads displaying three different Rv antigens of Mycobacterium tuberculosis was evaluated. Polyester beads displaying the antigens Rv1626, Rv2032, Rv1789, respectively, were produced in an endotoxin-free Escherichia coli strain. Beads were formulated with the adjuvant DDA and subcutaneously administered to C57BL/6 mice. Cytokine responses were evaluated by CBA and antibody responses by ELISA. Specificity of the IgG response was assessed by immunoblotting cell lysates of the vaccine production strains using sera from the vaccinated mice. Mice vaccinated with beads displaying Rv1626 had significantly greater IgG1 responses compared to mice vaccinated with Rv1789 beads and greater IgG2 responses than the group vaccinated with Rv2032 beads (p<0.05). Immunoblotting of antisera from these mice indicated the antibody responses were Rv1626 antigen-specific and there was no detectable immune response to the polyester component of the vaccine. Overall, this study suggested that selected TB antigens derived from reverse vaccinology approaches can be displayed on polyester beads to produce antigen-specific immune responses potentially relevant to the prevention of TB.

New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

Efficacy of Dry Heat Treatment against Clostridioides difficile Spores and Mycobacterium tuberculosis on Filtering Facepiece Respirators.

The COVID-19 pandemic has required novel solutions, including heat disinfection of personal protective equipment (PPE) for potential reuse to ensure availability for healthcare and other frontline workers. Understanding the efficacy of such methods on pathogens other than SARS-CoV-2 that may be present on PPE in healthcare settings is key to worker safety, as some pathogenic bacteria are more heat resistant than SARS-CoV-2. We assessed the efficacy of dry heat treatment against Clostridioides difficile spores and Mycobacterium tuberculosis (M. tb) on filtering facepiece respirator (FFR) coupons in two inoculums. Soil load (mimicking respiratory secretions) and deionized water was used for C. difficile, whereas, soil load and PBS and Tween mixture was used for M. tb. Dry heat treatment at 85 °C for 240 min resulted in a reduction equivalent to 6.0-log10 CFU and 7.3-log10 CFU in C. difficile spores inoculated in soil load and deionized water, respectively. Conversely, treatment at 75 °C for 240 min led to 4.6-log10 CFU reductions in both soil load and deionized water. C. difficile inactivation was higher by >1.5-log10 CFU in deionized water as compared to soil load (p < 0.0001), indicating the latter has a protective effect on bacterial spore inactivation at 85 °C. For M. tb, heat treatment at 75 °C for 90 min and 85 °C for 30 min led to 8-log10 reduction with or without soil load. Heat treatment near the estimated maximal operating temperatures of FFR materials (which would readily eliminate SARS-CoV-2) did not achieve complete inactivation of C. difficile spores but was successful against M. tb. The clinical relevance of surviving C. difficile spores when subjected to heat treatment remains unclear. Given this, any disinfection method of PPE for potential reuse must ensure the discarding of any PPE, potentially contaminated with C. difficile spores, to ensure the safety of healthcare workers.

Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Production of a particulate hepatitis C vaccine candidate by an engineered Lactococcus lactis strain.

Vaccine delivery systems based on display of antigens on bioengineered bacterial polyester inclusions can stimulate cellular immune responses. The food-grade Gram-positive bacterium Lactococcus lactis was engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which abundantly displayed the hepatitis C virus core (HCc) antigen. In mice, the immune response induced by this antigen delivery system was compared to that induced by vaccination with HCc antigen displayed on PHB beads produced in Escherichia coli, to PHB beads without antigen produced in L. lactis or E. coli, or directly to the recombinant HCc protein. Vaccination site lesions were minimal in all mice vaccinated with HCc PHB beads or recombinant protein, all mixed in the oil-in-water adjuvant Emulsigen, while vaccination with the recombinant protein in complete Freund's adjuvant produced a marked inflammatory reaction at the vaccination site. Vaccination with the PHB beads produced in L. lactis and displaying HCc antigen produced antigen-specific cellular immune responses with significant release of gamma interferon (IFN-γ) and interleukin-17A (IL-17A) from splenocyte cultures and no significant antigen-specific serum antibody, while the PHB beads displaying HCc but produced in E. coli released IFN-γ and IL-17A as well as the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 and low levels of IgG2c antibody. In contrast, recombinant HCc antigen in Emulsigen produced a diverse cytokine response and a strong IgG1 antibody response. Overall it was shown that L. lactis can be used to produce immunogenic PHB beads displaying viral antigens, making the beads suitable for vaccination against viral infections.

Immune response to allogeneic equine mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration. METHODS: Bone marrow was harvested from Connemara (n = 1), Standardbred (n = 6), and Thoroughbred (n = 3) horses. MSCs were grouped by their level of expression of major histocompatibility factor II (MHC II). MSCs were then sub-grouped by those MSCs derived from universal blood donor horses. MSCs were isolated and cultured using media containing fetal bovine serum until adequate numbers were acquired. The MSCs were cultured in xenogen-free media for 48 h prior to use and during all assays. Autologous and allogeneic MSCs were then directly co-cultured with responder leukocytes from the Connemara horse in varying concentrations of MSCs to leukocytes (1:1, 1:10, and 1:100). MSCs were also cultured with complement present and heat-inactivated complement to determine whether complement alone would decrease MSC viability. MSCs underwent haplotyping of their equine leukocyte antigen (ELA) to determine whether the MHC factors were matched or mismatched between the donor MSCs and the responder leukocytes. RESULTS: All allogeneic MSCs were found to be ELA mismatched with the responder leukocytes. MHC II-low and universal blood donor MSCs caused no peripheral blood mononuclear cell (PBMC) proliferation, no increase in B cells, and no activation of CD8 lymphocytes. Universal blood donor MSCs stimulated a significant increase in the number of T regulatory cells. Neutrophil interaction with MSCs showed that universal blood donor and MHC II-high allogeneic MSCs at the 6 h time point in co-culture caused greater neutrophil activation than the other co-culture groups. Complement-mediated cytotoxicity did not consistently cause MSC death in cultures with active complement as compared to those with inactivated complement. Gene expression assays revealed that the universal blood donor group and the MHC II-low MSCs were more metabolically active both in the anabolic and catabolic gene categories when cultured with allogeneic lymphocytes as compared to the other co-cultures. These upregulated genes included CD59, FGF-2, HGF, IDO, IL-10, IL-RA, IL-2, SOX2, TGF-ß1, ADAMSTS-4, ADAMSTS-5, CCL2, CXCLB/IL-8, IFNγ, IL-1ß, and TNFα. CONCLUSIONS: MHC II-low MSCs are the most appropriate type of allogeneic MSC to prevent activation of the innate and cell-mediated component of the adaptive immune systems and have increased gene expression as compared to other allogeneic MSCs.

CellTrace Violet™ inhibits equine lymphocyte proliferation.

CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than 90 % for both stained and unstained cells. Based on these data, CellTrace Violet™ is not recommended for the assessment of lymphocyte proliferation in equine cells. The mechanism of inhibition of equine lymphocyte proliferation by CellTrace Violet™ is unknown.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786 as a fusion ('MAP fusion protein particle'). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Bacterial polyester inclusions engineered to display vaccine candidate antigens for use as a novel class of safe and efficient vaccine delivery agents.

Bioengineered bacterial polyester inclusions have the potential to be used as a vaccine delivery system. The biopolyester beads were engineered to display a fusion protein of the polyester synthase PhaC and the two key antigens involved in immune response to the infectious agent that causes tuberculosis, Mycobacterium tuberculosis, notably antigen 85A (Ag85A) and the 6-kDa early secreted antigenic target (ESAT-6) from Mycobacterium tuberculosis. Polyester beads displaying the respective fusion protein at a high density were successfully produced (henceforth called Ag85A-ESAT-6 beads) by recombinant Escherichia coli. The ability of the Ag85A-ESAT-6 beads to enhance mouse immunity to the displayed antigens was investigated. The beads were not toxic to the animals, as determined by weight gain and absence of lesions at the inoculation site in immunized animals. In vivo injection of the Ag85A-ESAT-6 beads in mice induced significant humoral and cell-mediated immune responses to both Ag85A and ESAT-6. Vaccination with Ag85A-ESAT-6 beads was efficient at stimulating immunity on their own, and this ability was enhanced by administration of the beads in an oil-in-water emulsion. In addition, vaccination with the Ag85A-ESAT-6 beads induced significantly stronger humoral and cell-mediated immune responses than vaccination with an equivalent dose of the fusion protein Ag85A-ESAT-6 alone. The immune response induced by the beads was of a mixed Th1/Th2 nature, as assessed from the induction of the cytokine gamma interferon (Th1 immune response) and increased levels of immunoglobulin G1 (Th2 immune response). Hence, engineered biopolyester beads displaying foreign antigens represent a new class of versatile, safe, and biocompatible vaccines.

Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression.

BACKGROUND: As the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors. METHODS: The bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs. FINDINGS: There were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18. CONCLUSIONS: Universal blood donor- type Standardbred MSCs from passages 2-4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.

Phosphatidylinositol mannosides are efficient mucosal adjuvants.

The development of defined sub-unit vaccines requires the inclusion in the vaccine of an immunological adjuvant. The most important property of adjuvants for vaccines aimed at inducing optimal protection against intracellular bacteria such as Mycobacterium tuberculosis or M. bovis is the ability to enhance cell-mediated immunity, specifically Th1 responses. In this paper, we describe a system where transgenic mice expressing a high proportion of T cells specific for an ovalbumin (OVA) peptide are used to assess the ability of a novel class of adjuvants to positively modulate cell-mediated immune responses. Defined fractions containing purified native or synthetic phosphatidylinositol mannosides (PIMs) from mycobacteria were assessed for their adjuvant activities in response to the model antigen (OVA). Purified PIM preparations given to mice with OVA by the subcutaneous route were shown to elicit an enhanced release of interferon-gamma (IFN-gamma) in cellular responses to OVA peptide in vitro. Very little interleukin-4 (IL-4) was released by cells from mice immunized with PIMs and OVA, whereas cells from animals immunized with complete Freund's adjuvant (CFA) and OVA released IL-4 as well as IFN-gamma. Synthetic preparations of PIM2 and PIM4 also acted as adjuvants in the mouse model studied. In addition, PIM preparations were shown to generate an efficient cell-mediated immune response to OVA, when the antigen/adjuvant preparations were administered via the oral route or intranasal route. PIM preparations elicited substantial release of interleukin-12 (IL-12) from dendritic cells (DCs). These data suggest that purified or synthetic PIMs act as adjuvants when administered at mucosal surfaces and represent a new class of adjuvants for mucosal immunization against intracellular pathogens.

Bovine natural killer cells restrict the replication of Mycobacterium bovis in bovine macrophages and enhance IL-12 release by infected macrophages.

In this contribution, the impact of bovine natural killer (NK) cells on resistance to bovine tuberculosis was studied, using a monoclonal antibody against bovine NKp46. NK cells cultured with M. bovis-infected macrophages, but not control uninfected macrophages, proliferated and released IFN-gamma. Blood monocyte-derived macrophages were infected with virulent M. bovis, and growth of intra-macrophage bacteria was monitored by incorporation of tritiated uracil. Co-culturing infected macrophages with autologous NK cells significantly reduced the intracellular bacterial growth. Stimulation of NK cells with interleukin-2 (IL-2) enhanced further the capacity of these cells to reduce M. bovis replication in infected macrophages. NK cells from both BCG vaccinated and unvaccinated animals mediated this intra-macrophage growth restriction at similar levels. The ability of NK cells to reduce bacterial growth was independent of the release of IFN-gamma, as blocking IFN-gamma with an antibody in vitro did not affect intra-macrophage bacterial growth. NK cells reduced bacterial growth and also increased macrophage release of interleukin-12 (IL-12) and nitric oxide (NO) production by M. bovis-infected macrophages. Neutralizing NO production by macrophages in vitro with mono-methyl-l-arginine (MMLA) did not abrogate the ability of NK cells to decrease bacterial growth in infected macrophages. Reduction of mycobacterial intra-macrophage growth by NK cells was dependent on direct contact between NK cells and infected macrophages. Supernatants from NK cells failed to impact significantly on M. bovis replication in infected macrophages. The reduction in bacterial growth in macrophages correlated with the induction of an apoptosis program in infected macrophages. Cell death occurred at a similar rate in infected macrophages, exposed to NK cells or not. We conclude that bovine NK cells are stimulated by and release IFN-gamma in response to infected cells and reduce M. bovis growth in infected macrophages by an unclear mechanism, and are potentially involved in innate resistance of cattle to tuberculosis.

Bioengineering a bacterial pathogen to assemble its own particulate vaccine capable of inducing cellular immunity.

Many bacterial pathogens naturally form cellular inclusions. Here the immunogenicity of polyhydroxyalkanoate (PHA) inclusions and their use as particulate vaccines delivering a range of host derived antigens was assessed. Our study showed that PHA inclusions of pathogenic Pseudomonas aeruginosa are immunogenic mediating a specific cell-mediated immune response. Protein engineering of the PHA inclusion forming enzyme by translational fusion of epitopes from vaccine candidates outer membrane proteins OprI, OprF, and AlgE mediated self-assembly of PHA inclusions coated by these selected antigens. Mice vaccinated with isolated PHA inclusions produced a Th1 type immune response characterized by antigen-specific production of IFN-γ and IgG2c isotype antibodies. This cell-mediated immune response was found to be associated with the production of functional antibodies reacting with cells of various P. aeruginosa strains as well as facilitating opsonophagocytic killing. This study showed that cellular inclusions of pathogenic bacteria are immunogenic and can be engineered to display selected antigens suitable to serve as particulate subunit vaccines against infectious diseases.

Immunological properties and protective efficacy of a single mycobacterial antigen displayed on polyhydroxybutyrate beads.

In 2015, there were an estimated 10.4 million new tuberculosis (TB) cases and 1.4 million deaths worldwide. Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the vaccine available against TB, but it is insufficient for global TB control. This study evaluated the immunogenicity of the Mycobacterium tuberculosis antigen Rv1626 in mice while assessing the effect of co-delivering either Cpe30 (immunostimulatory peptide), CS.T3378-395 (promiscuous T helper epitope) or flagellin (TLR5 agonist) or a combination of all three immunostimulatory agents. Rv1626 and the respective immunostimulatory proteins/peptides were co-displayed on polyhydroxybutyrate beads assembled inside an engineered endotoxin-free mutant of Escherichia coli. Mice vaccinated with these beads produced immune responses biased towards Th1-/Th17-type responses, but inclusion of Cpe30, CS.T3378-395 and flagellin did not enhance immunogenicity of the Rv1626 protein. This was confirmed in a M. bovis challenge experiment in mice, where Rv1626 beads reduced bacterial cell counts in the lungs by 0.48 log10 compared with the adjuvant alone control group. Co-delivery of immunostimulatory peptides did not further enhance protective immunity.

Tuberculin Skin Testing Boosts Interferon Gamma Responses to DIVA Reagents in Mycobacterium bovis-Infected Cattle.

Mycobacterium bovis BCG vaccination sensitizes cattle to bovine tuberculin, which compromises the use of the current bovine tuberculosis (TB) surveillance tests. Although the performance of a blood test (that utilizes antigens expressed by Mycobacterium bovis but not by BCG) capable of discriminating infected from vaccinated animals (DIVA interferon gamma test [DIT]) has been evaluated in naturally infected TB field reactors, there is a need to perform similar analysis in a BCG-vaccinated M. bovis-infected population. Furthermore, we explored different scenarios under which a DIT may be implemented alongside BCG vaccination: (i) serial testing to resolve potential false-positive skin test results or (ii) a standalone test to replace the single intradermal comparative cervical tuberculin (SICCT) skin test. Our results demonstrated significantly better relative test sensitivity when the DIT was evaluated in a serial test scenario. Direct comparison of pre- and post-skin test blood samples revealed that the SICCT test induced significant boosting of the gamma interferon response in M. bovis-infected animals to both the ESAT-6-CFP-10 and Rv3615c peptide cocktails that comprise the DIT, which persisted for the ESAT-6-CFP-10 reagent for at least 14 days. Importantly, no similar boosting effects were observed in noninfected BCG vaccinates, suggesting that DIVA blood testing after a recent skin test would have minimal impact on test specificity.

Self-Assembled Protein-Coated Polyhydroxyalkanoate Beads: Properties and Biomedical Applications.

Polyhydroxyalkanoates (PHAs) are biological polyesters that can be naturally produced by a range of bacteria as water-insoluble inclusions composed of a PHA core coated with PHA synthesis, structural, and regulatory proteins. These naturally self-assembling shell-core particles have been recently conceived as biomaterials that can be bioengineered as biologically active beads for medical applications. Protein engineering of PHA-associated proteins enabled the production of PHA-protein assemblies exhibiting biologically active protein-based functions relevant for applications as vaccines or diagnostics. Here we provide an overview of the recent advances in bioengineering of PHA particles toward the display of biomedically relevant protein functions such as selected disease-specific antigens as diagnostic tools or for the design of particulate subunit vaccines against infectious diseases such as tuberculosis, meningitis, pneumonia, and hepatitis C.

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows.

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.