Arlene I. Ramsingh

Genetics of coxsackievirus B cardiovirulence and inflammatory heart muscle disease

Coxsackieviruses B (CVBs) are etiological agents of human inflammatory myocardial disease. The genetics of the coxsackieviral virulence phenotype in mice are now beginning to be understood with the availability of infectious cDNA copies of CVB genomes. Investigations to date with CVB3 and CVB4 have shown that sites within a non-translated region and in the capsid proteins can affect the virulence phenotype. The relative importance of these sites to expression of the phenotype remains unclear.

Severity of disease induced by a pancreatropic Coxsackie B4 virus correlates with the H-2K q locus of the major histocompatibility complex

Coxsackie B viruses are known etiological agents of pancreatic diseases, including diabetes. The pathogenesis of these infections is influenced by both host and viral factors. In this report, we examined whether the outcome of Coxsackie B4 virus infection is dependent on the genes within the major histocompatibility complex (MHC). We generated a pancreatic variant, CB4-V and established an animal model system of pancreatitis with concurrent hypoglycemia in mice. Infection of various B10 H-2 congenic strains of mice revealed that the development of hypoglycemia with accompanying pancreatitis was independent of the MHC haplotype. However, the severity of the disease as monitored by the extent and duration of hypoglycemia and by mortality rate was found to be associated with the H-2 haplotype, specifically the H-2Kq locus. Pancreatic damage induced by CB4-V appeared to be both immune-mediated and viral-mediated. Histological examination of pancreatic tissue from infected B10 H-2 congenic mice revealed an association between acute destruction of the exocrine pancreas and lymphocytic infiltration. This infiltration may correlate with immune-mediated destruction of the infected pancreatic tissue. Since preferential replication of CB4-V was not observed in the most susceptible B10 mouse strain, direct viral destruction may not be the major mechanism of pancreatic injury.

Progression or Resolution of Coxsackievirus B4-Induced Pancreatitis: a Genomic Analysis

ABSTRACT Group B coxsackieviruses are associated with chronic inflammatory diseases of the pancreas, heart, and central nervous system. Chronic pancreatitis, which can develop from acute pancreatitis, is considered a premalignant disorder because it is a major risk factor for pancreatic cancer. To explore the genetic events underlying the progression of acute to chronic disease, a comparative analysis of global gene expression during coxsackievirus B4-induced acute and chronic pancreatitis was undertaken. A key feature of acute pancreatitis that resolved was tissue regeneration, which was accompanied by increased expression of genes involved in cell growth, inhibition of apoptosis, and embryogenesis and by increased division of acinar cells. Acute pancreatitis that progressed to chronic pancreatitis was characterized by lack of tissue repair, and the expression map highlighted genes involved in apoptosis, acinoductular metaplasia, remodeling of the extracellular matrix, and fibrosis. Furthermore, immune responses appeared skewed toward development of alternatively activated (M2) macrophages and T helper 2 (Th2) cells during disease that resolved and toward classically activated (M1) macrophages and Th1 cells during disease that progressed. Our hypothesis is that growth and differentiation signals coupled with the M2/Th2 milieu favor acinar cell proliferation, while diminished growth signals and the M1/Th1 milieu favor apoptosis of acinar cells and remodeling/proliferation of the extracellular matrix, resulting in fibrosis.

Spontaneous cytokine gene expression in normal guinea pig blood and tissues

The authors report, for the first time, the cloning, characterization and sequencing of guinea pig cDNAs for interleukin (IL)-2, IL-10, IL-12p40, and transforming growth factor beta (TGF-beta). Partial cDNAs for two additional cytokines, IL-1alpha and TNF-alpha, whose sequences are present in the GenEMBL database, were also cloned. The IL-10 clone is a full-length cDNA, while the remaining clones are partial cDNAs. The guinea pig cDNA sequences have high identity with their mouse and human counterparts. Northern blot analysis revealed that the guinea pig transcripts range in size from 1.0 kb to 2.2 kb. The constitutive expression of cytokines in two strains of guinea pig (C4D, Albany) that differ in susceptibility to infection with Treponema pallidum was examined. Since susceptibility to T. pallidum is also age dependent, both neonates and adults were examined. Spontaneous cytokine expression was examined in peripheral blood, skin, spleen, lymph node, brain, and peritoneal cells. In skin, lymph node, and peripheral blood, very low levels of IL-1alpha, IL-12p40, tumour necrosis factor alpha (TNF-alpha), and TGF-beta and moderate levels of IL-2 and IL-10 were observed. Cytokine gene expression was not observed in spleen and brain. Peritoneal cells expressed only TGF-beta. Age- and strain-associated differences were not observed, except for IL-12p40, which was elevated in guinea pigs resistant to T. pallidum infection (C4D neonates, Albany adults).

IL-10 is pathogenic during the development of coxsackievirus B4-induced chronic pancreatitis

Using a mouse model of coxsackievirus B4 (CVB4-V)-induced chronic pancreatitis, we investigated whether cytokines are involved in the progression of acute disease to chronic inflammatory disease. We show that IL-10 contributed to the development of chronic pancreatitis since acute disease resolved when IL-10 was absent or when IL-10 signaling was disrupted. We explored the underlying mechanisms by which IL-10 affected disease progression, using a novel approach to assess immunological events occurring in situ. Multiple markers that define functional innate immune responses and functional T cell responses were monitored over the course of CVB4-V infection of wild-type and IL-10 knockout mice, using a multiplex transcriptional profiling approach. We show that high levels of IL-10 early during infection were associated with delayed innate and T cell responses. Furthermore, high IL-10 production correlated with altered kinetics of T regulatory responses indicating a disruption in the balance between effector and regulatory T cell responses.

Oral immunization with a live coxsackievirus/HIV recombinant induces gag p24-specific T cell responses.

Background The development of an HIV/AIDS vaccine has proven to be elusive. Because human vaccine trials have not yet demonstrated efficacy, new vaccine strategies are needed for the HIV vaccine pipeline. We have been developing a new HIV vaccine platform using a live enterovirus, coxsackievirus B4 (CVB4) vector. Enteroviruses are ideal candidates for development as a vaccine vector for oral delivery, because these viruses normally enter the body via the oral route and survive the acidic environment of the stomach. Methodology/Principal Findings We constructed a live coxsackievirus B4 recombinant, CVB4/p24(733), that expresses seventy-three amino acids of the gag p24 sequence (HXB2) and assessed T cell responses after immunization of mice. The CVB4 recombinant was physically stable, replication-competent, and genetically stable. Oral or intraperitoneal immunization with the recombinant resulted in strong systemic gag p24-specific T cell responses as determined by the IFN-γ ELISPOT assay and by multiparameter flow cytometry. Oral immunization with CVB4/p24(733) resulted in a short-lived, localized infection of the gut without systemic spread. Because coxsackieviruses are ubiquitous in the human population, we also evaluated whether the recombinant was able to induce gag p24-specific T cell responses in mice pre-immunized with the CVB4 vector. We showed that oral immunization with CVB4/p24(733) induced gag p24-specific immune responses in vector-immune mice. Conclusions/Significance The CVB4/p24(733) recombinant retained the physical and biological characteristics of the parental CVB4 vector. Oral immunization with the CVB4 recombinant was safe and resulted in the induction of systemic HIV-specific T cell responses. Furthermore, pre-existing vector immunity did not preclude the development of gag p24-specific T cell responses. As the search continues for new vaccine strategies, the present study suggests that live CVB4/HIV recombinants are potential new vaccine candidates for HIV.

Induction of mucosal HIV-specific B and T cell responses after oral immunization with live coxsackievirus B4 recombinants

Given the limited success of clinical HIV vaccine trials, new vaccine strategies are needed for the HIV pipeline. The present study explored the novel concept that a live enteric virus, with limited disease potential, is a suitable vaccine vector to elicit HIV-specific immune responses in the gut mucosa of immunized mice. Two coxsackievirus B4 (CVB4) vaccine vectors were designed to induce HIV-specific B or T cell responses. A B cell immunogen, CVB4/gp41(2F5), was constructed by expressing an epitope from the membrane proximal external region (MPER) of gp41 as a structural peptide within a surface loop of a capsid protein of CVB4. The T cell immunogen, CVB4/p24(73(3)), was constructed previously by expressing a gag p24 sequence as a non-structural peptide at the amino-terminus of the CVB4 polyprotein. The CVB4/gp41(2F5) recombinant was antigenic in mice and elicited anti-gp41 antibodies in both the mucosal and systemic compartments. The route of immunization affected the antibody response since oral delivery of CVB4/gp41(2F5) induced anti-gp41 antibodies in the mucosal but not in the systemic compartment while parenteral delivery induced anti-gp41 antibodies in both compartments. In contrast, oral immunization with CVB4/p24(73(3)) elicited both mucosal and systemic gag p24-specific T cell responses. Since coxsackieviruses are ubiquitous in the human population, a key question is whether pre-existing vector immunity will inhibit the ability of a CVB4-based vaccine to induce HIV-specific immune responses. We show that pre-existing vector immunity did not preclude the development of mucosal anti-gp41 antibodies or gag p24-specific T cell responses after oral immunization with the CVB4/HIV recombinants. We suggest that the CVB4/HIV recombinants have the potential to be a viable vaccine product because of ease of delivery, safety, immunogenicity, ease of large-scale production, and storage conditions requiring cold-chain temperatures provided by refrigeration.

Transcriptional dysregulation of inflammatory/immune pathways after active vaccination against Huntington's disease

Immunotherapy, both active and passive, is increasingly recognized as a powerful approach to a wide range of diseases, including Alzheimers and Parkinsons. Huntingtons disease (HD), an autosomal dominant disorder triggered by misfolding of huntingtin (HTT) protein with an expanded polyglutamine tract, could also benefit from this approach. Individuals can be identified genetically at the earliest stages of disease, and there may be particular benefits to a therapy that can target peripheral tissues in addition to brain. In this active vaccination study, we first examined safety and immunogenicity for a broad series of peptide, protein and DNA plasmid immunization protocols, using fragment (R6/1), and knock-in (zQ175) models. No safety issues were found. The strongest and most uniform immune response was to a combination of three non-overlapping HTT Exon1 coded peptides, conjugated to KLH, delivered with alum adjuvant. An N586-82Q plasmid, delivered via gene gun, also showed ELISA responses, mainly in the zQ175 strain, but with more variability, and less robust responses in HD compared with wild-type controls. Transcriptome profiling of spleens from the triple peptide-immunized cohort showed substantial HD-specific differences including differential activation of genes associated with innate immune responses, absence of negative feedback control of gene expression by regulators, a temporal dysregulation of innate immune responses and transcriptional repression of genes associated with memory T cell responses. These studies highlight critical issues for immunotherapy and HD disease management in general.

Dynamics of molecular responses to coxsackievirus B4 infection differentiate between resolution and progression of acute pancreatitis.

A coxsackievirus B4 induces acute pancreatitis with different outcomes. The study utilized a systems biology approach to identify molecular immune responses that differentiate between disease resolution and disease progression. The data establish a temporal pattern of host responses that differentiate the resolution of acute pancreatitis from the progression to chronic pancreatitis. A group of twenty-five genes exhibited characteristic expression profiles that were observed during the development of chronic pancreatitis but not during the resolution of disease. We postulate that the temporal dynamics of the twenty-five genes influence the development of pathogenic immune responses associated with chronic pancreatitis. Furthermore, a subset of eleven genes exhibited increased expression as viral titers waned. Of the eleven gene products, five are secreted molecules, TNF-α, IFN-γ, CXCL10, IL-10, and IL-22b, and represent novel potential therapeutic targets since they can be readily modulated with antibodies against the specific cytokine/chemokine or with antibodies against the corresponding receptors.

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation

A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.