Zebrafish as a model for infectious disease and immune function

The zebrafish, Danio rerio, has come to the forefront of biomedical research as a powerful model for the study of development, neurobiology, and genetics of humans. In recent years, use of the zebrafish system has extended into studies in behaviour, immunology and toxicology, retaining the concept that it will serve as a model for human disease. As one of the most thoroughly studied teleosts, with a wealth of genetic and genomic information available, the zebrafish is now being considered as a model for pathogen studies in finfishes. Its genome is currently being sequenced and annotated, and gene microarrays and insertional mutants are commercially available. The use of gene-specific knockdown of translation through morpholino oligonucleotides is widespread. As a result, several laboratories have developed bacterial and viral disease models with the zebrafish to study immune responses to infection. Although many of the zebrafish pathogen models were developed to address human infectious disease, the results of these studies should provide important clues for the development of effective vaccines and prophylactic measures against bacterial and viral pathogens in economically important fishes. In this review, the capabilities and potential of the zebrafish model system will be discussed and an overview of information on zebrafish infectious disease models will be presented.     

The function of dog models in developing gene therapy strategies for human health

The domestic dog is of great benefit to humankind, not only through companionship and working activities cultivated through domestication and selective breeding, but also as a model for biomedical research. Many single-gene traits have been well-characterized at the genomic level, and recent advances in whole-genome association studies will allow for better understanding of complex, multigenic hereditary diseases. Additionally, the dog serves as an invaluable large animal model for assessment of novel therapeutic agents. Thus, the dog has filled a crucial step in the translation of basic research to new treatment regimens for various human diseases. Four well-characterized diseases in canine models are discussed as they relate to other animal model availability, novel therapeutic approach, and extrapolation to human gene therapy trials.     

The Pekin duck programmed death ligand-2: cDNA cloning,genomic structure,molecular characterization and expression analysis

Programmed death-1 (PD-1), upon engagement by its ligands, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), provides signals that attenuate adaptive immune responses. Here we describe the identification of the Pekin duck PD-L2 (duPD-L2) and its gene structure. The duPD-L2 cDNA encodes a 321 amino acid protein that has an amino acid identity of 76% and 35% with chicken and human PD-L2, respectively. Mapping of the duPD-L2 cDNA with duck genomic sequences revealed an exonic structure similar to that of the human Pdcd1lg2 gene. Homology modelling of the duPD-L2 protein was compatible with the murine PD-L2 ectodomain structure. Residues known to be important for PD-1 receptor binding of murine PD-L2 were mostly conserved in duPD-L2 within sheets A and G and partially conserved within sheets C and F. DuPD-L2 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung, spleen, cloaca, bursa, cecal tonsil, duodenum and very low levels of expression in muscle, kidney and brain. Lipopolysaccharide treatment of adherent duck PBMC upregulated duPD-L2 mRNA expression. Our work shows evolutionary conservation of the PD-L2 ectodomain structure and residues important for PD-1 binding in vertebrates including fish. The information provided will be useful for further investigation of the role of duPD-L2 in the regulation of duck adaptive immunity and exploration of PD-1-targeted immunotherapies in the duck hepatitis B infection model.     

Adenoviral transduction of melanoma cells with B7-1: antitumor immunity and immunosuppressive factors

 Previous studies in experimental models have demonstrated that the transduction of human or murine melanoma cells with the co-stimulatory B7-1 molecule induces effective antitumor immune responses. In order to develop B7-1 gene transfer as a therapeutic tool in the clinical management of melanoma, efficient means of in vivo gene transfer must be used. To this end we evaluated in vitro and in vivo immune responses associated with adenoviral transduction of murine and human melanoma cells with B7-1. Adenovirus-mediated transduction of human and murine melanoma cells with B7-1 leads to high-level transgene expression in vitro and in vivo and does not affect MHC class I and II expression. Adenovirus-delivered B7-1 induced antitumor immune responses, on the basis of observations that human melanoma cells transduced to express human B7-1 were able to co-stimulate allogeneic and autologous T cells to proliferate and that murine melanoma K1735 cells transduced to express murine B7-1 were rejected by syngeneic, immunocompetent mice. By contrast, intratumoral injection of an adenovirus encoding murine B7-1 failed to eliminate established murine melanoma (K1735) despite high-level transgene expression in tumor cells. Potent T cell inhibitory factor(s) secreted by both K1735 cells and select human melanoma cells may contribute to the failure to achieve protection in this setting. Thus, immune inhibitory melanoma-derived factors need to be taken into account when considering the clinical use of B7-1 immunotherapy. Received: 21 December 1997 / Accepted: 16 March 1998     

Experimental mouse tumour models: what can be learnt about human cancer immunology?

The recent demonstration that cancer immunotherapy extends patient survival has reinvigorated interest in elucidating the role of immunity in tumour pathogenesis. Experimental mouse tumour models have provided key mechanistic insights into host antitumour immune responses, and these have guided the development of novel treatment strategies. To accelerate the translation of these findings into clinical benefits, investigators need to gain a better understanding of the strengths and limitations of mouse model systems as tools for deciphering human antitumour immune responses.     

Polynucleotide vaccines: potential for inducing immunity in animals.

Polynucleotide immunization has been described as the Third Revolution in Vaccinology. Early studies suggest the potential benefits of this form of immunization including: long-lived immunity, a broad-spectrum of immune responses (both cell mediated immunity, and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using a murine model, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to mucosal surfaces have been attempted and are described. Expansion of studies in various species, using natural models, should be extremely helpful in demonstrating the universality of this approach to immunization and more importantly, accurately identify parameters that are critical for the development of protective immunity.     

Comparative analysis of immune responses to Mycobacterium abscessus infection and its antigens in two murine models

Mycobacterium abscessus has been identified as an emerging pulmonary pathogen in humans. Because little is known regarding immune responses elicited by M. abscessus or its antigens, immunological responses were studied in two murine models subjected to intravenous (high-dose or systemic infection) or pulmonary (low-dose or local infection) inoculation with M. abscessus ATCC 19977. An overall comparison between the two models showed similar patterns of bacterial survival and host immune responses. The colonization of M. abscessus was the highest at 5 days post-infection (dpi) and its elimination was positively correlated with cell-mediated immunity in both challenges. However, an inverse relationship was observed between progressive inflammation and mycobacterial colonization levels in mice infected with a high dose at 14 dpi. Regarding antigens, culture filtrate (CF) of M. abscessus strongly induced IFN-γ secretion, whereas cellular extract (CE) antigen elicited strong antibody responses. The antibody response to M. abscessus antigens in mice subjected to low-dose infection increased when the cellular immune response decreased over 14 dpi. However, the antibody response for the high-dose infection increased promptly after the infection. In comparison of cytokine expression in lung homogenates after M. abscessus infection, Thl and Th2 cytokines increased simultaneously in the high-dose infection, whereas only cell-mediated immunity developed in the low-dose pulmonary infection. These findings not only enhance our understanding of the immune response to M. abscessus infection according to systemic or pulmonary infection, but may also aid in immunological diagnosis and vaccine development. M. abscessus, murine infection model, immune response, antigens, cytokines     

Phenotypic switching and its implications for the pathogenesis of Cryptococcus neoformans

Phenotypic switching has been described in several strains of Cryptococcus neoformans. It occurs in vivo during chronic infection and is associated with differential gene expression and changes in virulence. The switch involves changes in the polysaccharide capsule and cell wall that affect the yeast's ability to resist phagocytosis. In addition, the phenotypic switch variants elicit qualitatively different inflammatory responses in the host. The host's immune response ultimately affects selection of the switch variants in animal models of chronic cryptococcosis. The biological relevance of phenotypic switching is demonstrated in several murine infection models and further underlines the importance of phenotypic switching in the setting of human disease. This includes the association of switching and poor outcome in chronic infection, the ability of the mucoid variant of strain RC-2 (RC-2 MC) but not the smooth variant (RC-2 SM) to promote increased intracranial pressure in a rat model, and lastly the observation that antifungal interventions can promote the selection of more virulent switch variants during chronic murine infection.     

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.     

Influenza 2009 pandemic: Cellular immunemediated surveillance modulated by TH17 & Tregs

Influenza A virus is a serious public health threat. Most recently the 2009/H1N1 pandemic virus had an inherent ability to evade the host's immune surveillance through genetic drift, shift, and genomic reassortment. Immune characterization of 2009/H1N1 utilized monoclonal antibodies, neutralizing sera, and proteomics. Increased age may have provided some degree of immunity, but vaccines against seasonal influenza viruses seldom yield cross-reactive immunity, exemplified by 2009/H1N1. Nonetheless, about 33% of individuals, over the age of 60, had cross-reactive neutralizing antibodies against 2009/H1N1, whereas only 6-9% young adults had these antibodies. Children characteristically had no detectable immunity against 2009/H1N1. Taken together, these observations suggest some degree of immune transference with at least certain strains of virus that have afflicted the human population in past decades. Because internal influenza proteins may exhibit less antigenic variation, it is possible that prior exposure to diverse strains of influenza virus provide some immunity to novel strains, including the recent pandemic strain (swine-avian A/H1N1). Current trends in immunological studies - specifically the modulation of cellular immune surveillance provided by TH17 and Tregs - also support the need for additional proteomic research for characterizing novel translational evidence-based treatment interventions based on cytokine function to help defeat the virus. Timely and critical research must characterize the impact of genetics and epigenetics of oral and systemic host immune surveillance responses to influenza A virus. The continued development and application of proteomics and gene expression across viral strains and human tissues increases our ability to combat the spread of influenza epidemics and pandemics.     

The construction of transgenic and gene knockout/knockin mouse models of human disease

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.     

Transgene Regulation Using the Tetracycline-Inducible TetR-KRAB System after AAV-Mediated Gene Transfer in Rodents and Nonhuman Primates

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.     

An MVA vaccine overcomes tolerance to human p53 in mice and humans

Background The cellular regulatory protein p53 is overexpressed by almost 50% of all malignancies making it an attractive target for a vaccine approach to cancer. A number of immunotherapy approaches targeting p53 have been evaluated successfully in murine models, but translation of these preclinical findings to the clinic has been unsuccessful. Prior studies in our laboratory employing murine models demonstrated that a modified vaccinia virus Ankara (MVA) vaccine expressing murine p53 could stimulate p53 specific immunity. Systemic administration of the MVA vaccine was able to effect the rejection of established tumors. To better understand the immunologic mechanisms that underlie the vaccine function of human p53, we utilized a murine model in which the murine germ line copy of p53 was replaced with a modified human one. These mice, referred to as Hupki, were evaluated as a tolerant model to explore the capacity of MVA expressing human p53 to overcome tolerance and reject human p53-expressing tumors. Results MVAp53 immunization of Hupki mice resulted in the generation of p53-specific CD8⁺ T cells and the rejection of a highly aggressive murine mammary carcinoma cell line 4T1(H-2d) transfected with human p53 (4T1p53). An immunologic correlate of tumor protection was evaluated utilizing an overlapping peptide library spanning the full length of human p53. This reagent was also used in combination with MVAp53 to stimulate p53-specific CD8⁺ T cell responses in cancer patients. Conclusion These studies demonstrate the potential of MVAp53 to overcome tolerance to p53 for cancer immunotherapy.     

GM-CSF transgene-based adjuvant allows the establishment of protective mucosal immunity following vaccination with inactivated Chlamydia trachomatis

Cellular and humoral immune responses induced following murine Chlamydia trachomatis infection confer almost sterile protection against homologous reinfection. On the other hand, immunization with inactivated organism induces little protective immunity in this model system. The underlying mechanism(s) that determines such divergent outcome remains unclear, but elucidating the mechanism will probably be important for chlamydial vaccine development. One of the distinct differences between the two forms of immunization is that chlamydia replication in epithelial cells causes the secretion of a variety of proinflammatory cytokines and chemokines, such as GM-CSF, that may mobilize and mature dendritic cells and thereby enhance the induction of protective immunity. Using a murine model of C. trachomatis mouse pneumonitis lung infection and intrapulmonary adenoviral GM-CSF transfection, we demonstrate that the expression of GM-CSF in the airway compartment significantly enhanced systemic Th1 cellular and local IgA immune responses following immunization with inactivated organisms. Importantly, immunized mice had significantly reduced growth of chlamydia and exhibited less severe pulmonary inflammation following challenge infection. The site of GM-CSF transfection proved important, since mice immunized with inactivated organisms after GM-CSF gene transfer by the i.p. route exhibited little protection against pulmonary challenge, although i.p. immunization generated significant levels of systemic Th1 immune responses. The obvious difference between i.p. and intrapulmonary immunization was the absence of lung IgA responses following i.p. vaccination. In aggregate, the findings demonstrate that the local cytokine environment is critical to the induction of protective immunity following chlamydial vaccination and that GM-CSF may be a useful adjuvant for a chlamydial vaccine.