Janice J. Endsley

Maternal Antibody Blocks Humoral but Not T Cell Responses to BVDV

Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.

Characterization of Bovine Homologues of Granulysin and NK-lysin

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3+ T cells, CD4+ T cells, CD8+ T cells, WC1+ γδ T cells, and PBMC depleted of CD3+ T cells, but was absent in CD21+ cells and CD14+ cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.

A Humanized Mouse Model of Tuberculosis

Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γc null mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34+ fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45+) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2–8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.

Bovine natural killer cells acquire cytotoxic/effector activity following activation with IL‐12/15 and reduce Mycobacterium bovis BCG in infected macrophages

Bovine natural killer (NK) cells were recently identified by positive selection of a NK cell‐activating receptor p46 (NKp46)+ CD3– lymphocyte population, which expresses CD25 and CD8 and lyses tumor cell lines following stimulation with recombinant interleukin‐2. In the current work, we characterize the cytotoxic/effector potential of a CD3–CD8–CD11b– population isolated through negative selection of bovine peripheral blood leukocytes. This population is CD25loCD62hi when isolated and becomes CD25hiCD62Llo following cytokine stimulation. Activated bovine NK cells increase expression of granulysin, interferon‐γ, and perforin and have cytotoxic activity against human tumor cells and Mycobacterium bovis bacillus Calmette‐Guerin‐infected alveolar and monocyte‐derived macrophages. Expression of a bovine homologue of the CD56 neural adhesion molecule expressed by human NK cells was detected in mRNA from brain tissue but was not detected in peripheral blood mononuclear cells or purified NK cell mRNA. Analysis of mRNA from nonstimulated peripheral blood NK cells demonstrates the constitutive expression of homologues of human NK receptors NKp46, CD244, and CD94 and the granule proteins granulysin and perforin. Phorbol ester‐stimulated CD8+ T cells also expressed CD244 and CD94, and CD4+ T cells expressed CD94. These NK cell receptors bearing T lymphocytes may represent memory subsets characterized in humans. The results of these studies demonstrate that bovine NK cells may play an important role in the innate immune responses of cattle.

Induction of granulysin in CD8+ T cells by IL-21 and IL-15 is suppressed by human immunodeficiency virus-1

Immunosuppression following infection with HIV‐1 predisposes patients to a myriad of opportunistic pathogens, one of the most important of which is Mtb. Granulysin, expressed by NK cells and CTL, exhibits potent antimicrobial activity against Mtb and several other opportunistic pathogens associated with HIV‐1 infection. The immune signals that promote granulysin expression in human CTL are not fully understood. Using primary human CD8+ T cells, in this study, we identify IL‐21 as a strong inducer of granulysin, demonstrate that IL‐21 and IL‐15 activate granulysin expression within CD8+ CD45RO+ T cells, and establish a role for Jak/STAT signaling in the regulation of granulysin within CD8+ T cells. We show that infection of PBMC from healthy donors in vitro with HIV‐1 suppresses granulysin expression by CD8+ T cells, concomitant with reduced p‐STAT3 and p‐STAT5, following activation with IL‐15 and IL‐21. Of note, simultaneous signaling through IL‐15 and IL‐21 could partially overcome the immunosuppressive effects of HIV‐1 on granulysin expression by CD8+ T cells. These results suggest that HIV‐1 infection of PBMC may reduce the antimicrobial profile of activated CD8+ T cells by disrupting signaling events that are critical for the induction of granulysin. Understanding the effects of HIV‐1 on CD8+ T cell activation is essential to understanding the physiological basis for inadequate cytotoxic lymphocyte activity in HIV+ patients and for informed guidance of cytokine‐based therapy to restore T cell function.

Induction of T lymphocytes specific for bovine viral diarrhea virus in calves with maternal antibody.

Passive antibody to bovine viral diarrhea virus (BVDV) acquired through colostrum intake may interfere with the development of a protective immune response by calves to this virus. The objective of this study was to determine if calves, with a high level of maternal antibody to bovine viral diarrhea virus (BVDV), develop CD4(+), CD8(+), or gammadelta T lymphocyte responses to BVDV in the absence of a measurable humoral immune response. Colostrum or milk replacer fed calves were challenged with virulent BVDV at 2-5 weeks of age and/or after maternal antibody had waned. Calves exposed to BVDV while passive antibody levels were high did not mount a measurable humoral immune response to BVDV. However, compared to nonexposed animals, these animals had CD4(+), CD8(+), and gammadelta T lymphocytes that were activated by BVDV after exposure to in vitro BVDV. The production of IFNgamma by lymphocytes after in vitro BVDV exposure was also much greater in lymphocytes from calves exposed to BVDV in the presence of maternal antibody compared to the nonexposed calves. These data indicate that calves exposed to BVDV while maternal antibody levels are high can develop antigen specific CD4(+), CD8(+), and gammadelta T lymphocytes in the absence of an active antibody response. A manuscript presented separately demonstrates that the calves with T lymphocytes specific for BVDV in this study were also protected from virulent BVDV genotype 2 challenge after maternal antibody became undetectable.

Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks

ABSTRACT We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutants growth dropped slightly during the late phase at 37°C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 105 to 5 × 105 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 108 CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

CXCL10 production by human monocytes in response to Leishmania braziliensis infection.

ABSTRACT Leishmania (subgenus Viannia) braziliensis is the causative agent of mucocutaneous leishmaniasis (ML) in South America, and ML is characterized by excessive T- and B-cell responses to the parasite. We speculate that the unbalanced production of inflammatory mediators in response to L. braziliensis infection contributes to cell recruitment and disease severity. To test this hypothesis, we first examined the response of peripheral blood mononuclear cells (PBMCs) from healthy volunteers to L. braziliensis infection. We observed that while L. braziliensis infection induced the production of chemokine (C-X-C motif) ligand 10 (CXCL10) and interleukin-10 (IL-10) in human PBMCs and macrophages (MΦs), enhanced expression of CXCL10 and its receptor, chemokine CXC receptor (CXCR3), was predominantly detected in CD14+ monocytes. The chemoattractant factors secreted by L. braziliensis-infected cells were highly efficient in recruiting uninfected PBMCs (predominantly CD14+ cells) through Transwell membranes. Serum samples from American tegumentary leishmaniasis (ATL) patients (especially the ML cases) had significantly higher levels of CXCL10, CCL4, and soluble tumor necrosis factor (TNF) receptor II (sTNFRII) than did those of control subjects. Our results suggest that, following L. braziliensis infection, the production of multiple inflammatory mediators by the host may contribute to disease severity by increasing cellular recruitment.

The calf model of immunity for development of a vaccine against tuberculosis.

Tuberculosis (TB) remains a major threat to public health. The identification of safe TB vaccine candidates beyond Mycobacterium bovis BCG, is an exciting prospect for control of human TB and necessary in the context of the human immunodeficiency virus (HIV) pandemic. Selection of vaccine candidates for human trials which are ultimately targeted for use in children less than 5 years of age or in newborns will require an animal model that closely approximates immune function and disease. We propose that the bovine neonate and adolescent is a robust animal model for preclinical safety and efficacy evaluation of TB candidate vaccines targeting this special human population. Parallel studies conducted in bovine neonates and non-human primates with a leading auxotrophic mutant with demonstrated efficacy/safety in a rodent TB model of TB demonstrated similar findings with respect to gross pathology scoring relative to BCG. The findings indicated more numerous and severe lesions in the lung in addition to higher levels of IFN-gamma producing cells. BCG vaccinates demonstrated higher levels of FoxP3 transcripts and lower levels of IL-4 mRNA.

Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection

Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens.