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In JoVE (1)
Other Publications (14)
- Journal of Medical Microbiology
- Viral Immunology
- Current Pharmaceutical Biotechnology
- Journal of Virology
- Genetic Vaccines and Therapy
- Microbial Pathogenesis
- Human Gene Therapy
- Animal Health Research Reviews / Conference of Research Workers in Animal Diseases
- Transactions of the Royal Society of Tropical Medicine and Hygiene
- Journal of Immune Based Therapies and Vaccines
- The Journal of Biological Chemistry
- Infection and Immunity
- The Biochemical Journal
- Journal of Bacteriology
Articles by Jerome Harms in JoVE
JoVE Immunology and Infection
Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells
1Pathology and Laboratory Medicine, University of Wisconsin-Madison, 2Pathobiological Sciences, University of Wisconsin-Madison
Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.
Other articles by Jerome Harms on PubMed
Induction of a Th1-type of Immune Response but Not Protective Immunity by Intramuscular DNA Immunisation with Brucella Abortus GroEL Heat-shock Gene
The immunogenicity and protective efficacy of a DNA vaccine encoding the GroEL heat-shock gene from Brucella abortus was tested in BALB/c mice immunised by intramuscular (i.m.) needle injection or epidermally by gene gun. The Brucella GroEL gene was amplified by PCR and cloned into two different mammalian expression vectors pCMV-link and pCMV-tPA. The D17 cell line was transfected with both constructs and GroEL transcripts were detected by Northern blot. To determine the level of protein synthesised, transfected cell lysates were then submitted to Western blot. The non-secreted form of the recombinant GroEL produced by the pCMV-link construct was detected in much greater amount than the secreted form of the protein produced by the pCMV-tPA construct. After immunisation, a strong anti-GroEL IgG response was detected in mice vaccinated by i.m. injection or gene gun only when the pCMV-link/ GroEL plasmid was used. Regarding the pattern of immune response induced, i.m. needle injection raised a predominantly Th1 response with mostly IgG2a-specific anti-GroEL and high levels of IFN-gamma produced by splenic T cells. Gene gun immunisation induced a ThO type of immune response in mice characterised by a high IgG1/IgG2a ratio, and IL-4 and interferon (IFN)-gamma production. Even though a distinct pattern of immune response was generated depending upon the immunisation route used, neither method engendered a significant level of protection with the GroEL DNA vaccine.
Encephalomyocarditis and Mengo Viruses Productively Infect Murine T-lymphocyte Cell Lines but Not Fresh Ex Vivo Derived T Lymphocytes
Encephalomyocarditis virus (EMCV) and Mengo virus are highly virulent murine cardioviruses that are found in abundant quantities in the spleen and lymph nodes after infection. T lymphocytes are pivotal mediators of humoral and cellular immunity against cardioviral challenge, and are highly suspect candidates of EMCV and Mengo virus infection. We found T lymphocyte-like cell lines CTLL-2, EL-4, LY1+2/9, and LBRM33 were susceptible to productive viral infection and exhibited cytopathology after infection with virulent EMCV-R or attenuated Mengo virus strains vMC0 and vMC24. Flow cytometric analysis demonstrated progressive intracellular accumulation of viral proteins, such as the replication-dependent 3D viral polymerase, in EL-4 cells during infection. Conversely, freshly isolated and mitogen-stimulated CD4+ and CD8+ T cells were resistant to productive infection with these viruses, exhibiting no viral-induced cytopathic effects or intracellular presence of viral proteins. These data indicate that although T-lymphocyte-like tumor cell lines are highly susceptible to viral infection and cytopathic effects, primary/freshly isolated T cells are resistant to infection by EMCV-R or Mengo virus.
Enhanced Efficacy of DNA Vaccines Against an Intracellular Bacterial Pathogen by Genetic Adjuvants
After 200 years of practice, vaccinology has proved to be very effective in preventing infectious diseases. However, several human and animal pathogens exist for which vaccines need to be improved or simply have not yet been discovered. The era of molecular genetic has given a new breath for vaccine development with the achievement of the "Third Generation of Vaccines": the DNA vaccine. In this article, we reviewed strategies that have been used to improve and modulate the immune response induced by DNA vaccines, using as a model the intracellular bacterial pathogen Brucella abortus. First, we described different approaches used to isolate and to identify genes that encode potential immunogens. Secondly, we reported the use of cytokine genes and genetic adjuvants that could improve the immunogenicity of target genes. And finally, we discussed the "Expression Library Immunization"-(ELI) strategy and the recent results obtained against Brucella abortus infection.
Bovine Herpesvirus Tegument Protein VP22 Enhances Thymidine Kinase/ganciclovir Suicide Gene Therapy for Neuroblastomas Compared to Herpes Simplex Virus VP22
Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.
Comparison of Bovine Leukemia Virus (BLV) and CMV Promoter-driven Reporter Gene Expression in BLV-infected and Non-infected Cells
BACKGROUND: Viral promoters are used in mammalian expression vectors because they generally have strong activity in a wide variety of cells of differing tissues and species. METHODS: The utility of the BLV LTR/promoter (BLVp) for use in mammalian expression vectors was investigated through direct comparison to the CMV promoter (CMVp). Promoter activity was measured using luciferase assays of cell lines from different tissues and species stably transduced with BLVp or CMVp driven luciferase vectors including D17, FLK, BL3.1 and primary bovine B cells. Cells were also modified through the addition of BLV Tax expression vectors and/or BLV infection as well as treatment with trichostatin A (TSA). RESULTS: Results indicate the BLV promoter, while having low basal activity compared to the CMV promoter, can be induced to high-levels of activity similar to the CMV promoter in all cells tested. Tax or BLV infection specifically enhanced BLVp activity with no effect on CMVp activity. In contrast, the non-specific activator, TSA, enhanced both BLVp and CMVp activity. CONCLUSION: Based on these data, we conclude the BLV promoter could be very useful for transgene expression in mammalian expression vectors.
The Role of Integrase/recombinase XerD and Monofunctional Biosynthesis Peptidoglycan Transglycosylase Genes in the Pathogenicity of Brucella Abortus Infection in Vitro and in Vivo
Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.
Bovine Herpesvirus VP22 Induces Apoptosis in Neuroblastoma Cells by Upregulating the Expression Ratio of Bax to Bcl-2
Herpesvirus tegument protein VP22 has been shown to have biotherapeutic potential in tumor gene therapy. Some studies indicate that VP22 may enhance the transfer efficiency of therapeutic proteins by delivering them to more cells while trafficking. Our previous study showed that bovine herpesvirus VP22 (BVP22) enhanced equine herpesvirus thymidine kinase-ganciclovir (Etk-GCV) suicide gene therapy by an unknown intracellular effect. In this study, the interaction between BVP22 and host tumor cells was studied in neuroblastoma NXS2 cells. Cell cycle analysis was performed to determine whether BVP22 possesses biotherapeutic potential by altering the cell cycle, making cells more sensitive to therapeutic genes. As a result, the cell cycle was not affected by the transfection of BVP22 into NXS2 cells. However, cytotoxicity induced by BVP22 was observed in NXS2 cells on the second and third days after transient transfection. Further, analyses of caspase-3 activity and apoptosis suggested that BVP22 induces apoptosis in host tumor cells by upregulating the expression ratio of Bax to Bcl-2.
Brucella: Functional Genomics and Host-pathogen Interactions
Brucellosis is a zoonotic disease caused by a number of Brucella species and is characterized by chronic macrophage infection. However, genes that may contribute to intracellular survival of the Brucella species are not well studied. This review presents, first, genomic islands that are present or absent in various Brucella species that may help establish Brucella infection and survival strategies. Second, the alteration in macrophage transcription by Brucella to permit its long-term survival within this hostile intracellular environment. A large number of macrophage gene transcripts are altered following Brucella infection indicating that Brucella is not a silent invader of host cells. Macrophage transcript levels associated with inflammation, apoptosis, signal transduction and vesicular intracellular trafficking are altered during Brucella infection, and likely contribute to intracellular survival of Brucella. Lastly, the host-pathogen interaction events associated with Brucella infection in living mice visualized in real-time using biophotonic imaging. Mice are often used to evaluate Brucella infections; however, Brucella dissemination and pathogenesis is poorly understood in mice. Biophotonic imaging of Brucella infections revealed sites of bacterial localization similar to human infections and different patterns of infection by attenuated or virulent Brucella.
TBK1 Does Not Play a Role in the Control of in Vitro Burkholderia Pseudomallei Growth
Burkholderia pseudomallei, the causative agent of melioidosis, is an important intracellular pathogen in tropical regions. TANK-binding kinase (TBK1), part of the pathway that induces transcription of Type I interferon genes, has been demonstrated to play an important role in controlling intracellular bacterial infections. To investigate the role of tbk1 in protecting against B. pseudomallei we developed tbk1-deficient cell lines by using shRNA for transient knockdown of the tbk1 gene in HeLa and RAW 264.7 cells. In tbk1-deficient RAW cells, the replication of invasive and non-invasive Escherichia coli was significantly increased at 48 h after infection compared with wild-type cells. The result was confirmed using Brucella melitensis in tbk1-deficient HeLa cells, which demonstrated a >1.5-2.0 log higher bacterial count at 6-48 h after infection compared to wild-type cells. By contrast, the growth of Burkholderia pseudomallei expressing either typical (A2) or atypical (G207) lipopolysaccharide was not significantly different between the tbk1-deficient and control cells. These results suggest that the tbk1 gene and its activation may be able to control invasive E. coli, non-invasive E. coli and B. melitensis growth but may not be able to control B. pseudomallei infection. The role of the tbk1 gene in proinflammatory cytokine induction and bacterial intracellular infection needs further investigation to identify mechanistic differences among the life cycles of various intracellular bacteria.
Evaluation of Recombinant Invasive, Non-pathogenic Eschericia Coli As a Vaccine Vector Against the Intracellular Pathogen, Brucella
ABSTRACT:
Brucella TIR Domain-containing Protein Mimics Properties of the Toll-like Receptor Adaptor Protein TIRAP
Toll-like receptors (TLRs) play essential roles in the activation of innate immune responses against microbial infections. TLRs and downstream adaptor molecules contain a conserved cytoplasmic TIR domain. TIRAP is a TIR domain-containing adaptor protein that recruits the signaling adaptor MyD88 to a subset of TLRs. Many pathogenic microorganisms subvert TLR signaling pathways to suppress host immune responses to benefit their survival and persistence. Brucella encodes a TIR domain-containing protein (TcpB) that inhibits TLR2- and TLR4-mediated NF-kappaB activation. Sequence analysis indicated a moderate level of similarity between TcpB and the TLR adaptor molecule TIRAP. We found that TcpB could efficiently block TIRAP-induced NF-kappaB activation. Subsequent studies revealed that by analogy to TIRAP, TcpB interacts with phosphoinositides through its N-terminal domain and colocalizes with the plasma membrane and components of the cytoskeleton. Our findings suggest that TcpB targets the TIRAP-mediated pathway to subvert TLR signaling. In vivo mouse studies indicated that TcpB-deficient Brucella is defective in systemic spread at the early stages of infection.
Discordant Brucella Melitensis Antigens Yield Cognate CD8+ T Cells in Vivo
Brucella spp. are intracellular bacteria that cause the most frequent zoonosis in the world. Although recent work has advanced the field of Brucella vaccine development, there remains no safe human vaccine. In order to produce a safe and effective human vaccine, the immune response to Brucella spp. requires greater understanding. Induction of Brucella-specific CD8+ T cells is considered an important aspect of the host response; however, the CD8+ T-cell response is not clearly defined. Discovering the epitope containing antigens recognized by Brucella-specific CD8+ T cells and correlating them with microarray data will aid in determining proteins critical for vaccine development that cover a kinetic continuum during infection. Developing tools to take advantage of the BALB/c mouse model of Brucella melitensis infection will help to clarify the correlates of immunity and improve the efficacy of this model. Two H-2(d) CD8+ T-cell epitopes have been characterized, and a group of immunogenic proteins have provoked gamma interferon production by CD8+ T cells. RYCINSASL and NGSSSMATV induced cognate CD8+ T cells after peptide immunization that showed specific killing in vivo. Importantly, we found by microarray analysis that the genes encoding these epitopes are differentially expressed following macrophage infection, further emphasizing that these discordant genes may play an important role in the pathogenesis of B. melitensis infection.
Modulation of Microtubule Dynamics by a TIR Domain Protein from the Intracellular Pathogen Brucella Melitensis
TIR (Toll/interleukin-1 receptor) domain-containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB (nuclear factor κB) activation and pro-inflammatory cytokine secretions mediated by TLR (Toll-like receptor) 2 and TLR4. In the present study, we have demonstrated that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation in a similar manner to paclitaxel. TcpB could efficiently inhibit nocodazole- or cold-induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain, and a point mutation affected the microtubule stabilization as well as the TLR-suppression properties of TcpB.
Brucella Melitensis Cyclic Di-GMP Phosphodiesterase BpdA Controls Expression of Flagellar Genes
Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.
