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Articles by Jeanon Smith in JoVE

 JoVE Immunology and Infection

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

1Experimental Pathology, University of Texas Medical Branch

JoVE 4429

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Other articles by Jeanon Smith on PubMed

Variability of Virus Attachment Patterns to Butterhead Lettuce

Enteric viruses account for most foodborne illness in the United States. The objective of this study was to determine whether the isoelectric point (pI) of viruses such as feline calicivirus (FCV), echovirus 11, and bacteriophages phiX174 and MS2 had any effect on their attachment to butterhead lettuce. The adsorption of virus particles to the lettuce was variable. Bacteriophage MS2 was the only virus that fit the current Derjaguin-Landau-Verway-Overbeek model of virus attachment. Echovirus 11 had the highest affinity to lettuce surface. Echovirus 11 appeared to exhibit reversible attachment above its pI, whereas below its pI strong adsorption was observed. Adsorption of FCV was at its maximum above its pI. Bacteriophage phiX174 exhibited the most complex adsorption pattern, with attachment occurring only at the pH extremes (pH 3.0 and 8.0). These results suggest the current model for virus adsorption to sediment does not adequately explain the attachment of virus to lettuce. Importantly, the results indirectly suggest that current sample processing methods to recover viruses from lettuce may differentially select for the recovery of only certain virus types.

Injectable Peramivir Mitigates Disease and Promotes Survival in Ferrets and Mice Infected with the Highly Virulent Influenza Virus, A/Vietnam/1203/04 (H5N1)

The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir-treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16-18 day period resulted from peramivir treatment, with improved survival of 40-64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets.

Novel Linear DNA Vaccines Induce Protective Immune Responses Against Lethal Infection with Influenza Virus Type A/H5N1

Vaccine development for possible influenza pandemics has been challenging. Conventional vaccines such as inactivated and live attenuated virus preparations are limited in terms of production speed and capacity. DNA vaccination has emerged as a potential alternative to conventional vaccines against influenza pandemics. In this study, we use a novel, cell-free DNA manufacturing process (synDNA) to produce prototype linear DNA vaccines against the influenza virus type A/H5N1. This synDNA process does not require bacterial fermentation, so it avoids the use of antibiotic resistance genes and other nucleic acid sequences unrelated to the antigen gene expression in the actual therapeutic DNA construct. The efficacy of various vaccines expressing the hemagglutinin and neuraminidase proteins (H5N1 synDNA), hemagglutinin alone (H5 synDNA) or neuraminidase alone (N1 synDNA) was evaluated in mice. Two of the constructs (H5 synDNA and H5N1 synDNA) induced a robust protective immune response with up to 93% of treated mice surviving a lethal challenge of a virulent influenza A/Vietnam/1203/04 H5N1 isolate. In combination with a potent biological activity and simplified production footprint, these characteristics make DNA vaccines prepared with our synDNA process highly suitable as alternatives to other vaccine preparations.

Pathogenesis of XJ and Romero Strains of Junin Virus in Two Strains of Guinea Pigs

Argentine hemorrhagic fever (AHF), a systemic infectious disease caused by infection with Junin virus, affects several organs, and patients can show hematologic, cardiovascular, renal, or neurologic symptoms. We compared the virulence of two Junin virus strains in inbred and outbred guinea pigs with the aim of characterizing this animal model better for future vaccine/antiviral efficacy studies. Our data indicate that this passage of the XJ strain is attenuated in guinea pigs. In contrast, the Romero strain is highly virulent in Strain 13 as well as in Hartley guinea pigs, resulting in systemic infection, thrombocytopenia, elevated aspartate aminotransferase levels, and ultimately, uniformly lethal disease. We detected viral antigen in formalin-fixed, paraffin-embedded tissues. Thus, both guinea pig strains are useful animal models for lethal Junin virus (Romero strain) infection and potentially can be used for preclinical trials in vaccine or antiviral drug development.

CD4+ T Cells Provide Protection Against Acute Lethal Encephalitis Caused by Venezuelan Equine Encephalitis Virus

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Superior Efficacy of a Recombinant Flagellin:H5N1 HA Globular Head Vaccine is Determined by the Placement of the Globular Head Within Flagellin

Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.

TC83 Replicon Vectored Vaccine Provides Protection Against Junin Virus in Guinea Pigs

Junin virus (JUNV) is the etiological agent of the potentially lethal, reemerging human disease, Argentine hemorrhagic fever (AHF). The mechanism of the disease development is not well understood and no antiviral therapy is available. Candid 1, a live-attenuated vaccine, has been developed by the US Army and is being used in the endemic area to prevent AHF. This vaccine is only approved for use in Argentina. In this study we have used the alphavirus-based approach to engineer a replicon system based on a human (United States Food and Drug Administration Investigational New Drug status) vaccine TC83 that express heterologous viral antigens, such as glycoproteins (GPC) of Junin virus (JUNV). Preclinical studies testing the immunogenicity and efficacy of TC83/GPC were performed in guinea pigs. A single dose of the live-attenuated alphavirus based vaccine expressing only GPC was immunogenic and provided partial protection, while a double dose of the same vaccine provided a complete protection against JUNV. This is the first scientific report to our knowledge that the immune response against GPC alone is sufficient to prevent lethal disease against JUNV in an animal model.

Mice Lacking Alpha/beta and Gamma Interferon Receptors Are Susceptible to Junin Virus Infection

Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease.

Prevention of Influenza Virus Shedding and Protection from Lethal H1N1 Challenge Using a Consensus 2009 H1N1 HA and NA Adenovirus Vector Vaccine

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.

Rapid, Non-invasive Imaging of Alphaviral Brain Infection: Reducing Animal Numbers and Morbidity to Identify Efficacy of Potential Vaccines and Antivirals

Rapid and accurate identification of disease progression are key factors in testing novel vaccines and antivirals against encephalitic alphaviruses. Typical efficacy studies utilize a large number of animals and severe morbidity or mortality as an endpoint. New technologies provide a means to reduce and refine the animal use as proposed in Hume's 3Rs (replacement, reduction, refinement) described by Russel and Burch. In vivo imaging systems (IVIS) and bioluminescent enzyme technologies accomplish the reduction of animal requirements while shortening the experimental time and improving the accuracy in localizing active virus replication. In the case of murine models of viral encephalitis in which central nervous system (CNS) viral invasion occurs rapidly but the disease development is relatively slow, we visualized the initial brain infection and enhance the data collection process required for efficacy studies on antivirals or vaccines that are aimed at preventing brain infection. Accordingly, we infected mice through intranasal inoculation with the genetically modified pathogen, Venezuelan equine encephalitis, which expresses a luciferase gene. In this study, we were able to identify the invasion of the CNS at least 3 days before any clinical signs of disease, allowing for reduction of animal morbidity providing a humane means of disease and vaccine research while obtaining scientific data accurately and more rapidly. Based on our data from the imaging model, we confirmed the usefulness of this technology in preclinical research by demonstrating the efficacy of Ampligen, a TLR-3 agonist, in preventing CNS invasion.

Neuropathology of H5N1 Virus Infection in Ferrets

Highly pathogenic H5N1 virus remains a potential threat to humans. Over 289 fatalities have been reported in WHO confirmed human cases since 2003, and lack of effective vaccines and early treatments contribute to increasing numbers of cases and fatalities. H5N1 encephalitis is a recognized cause of death in Vietnamese cases, and brain pathology is described in other human cases and naturally infected animals. However, neither pathogenesis of H5N1 viral infection in human brain nor post-infection effects in survivors have been fully investigated. We report the brain pathology in a ferret model for active infection and 18-day survival stages. This model closely resembles the infection pattern and progression in human cases of influenza A, and our report is the first description of brain pathology for longer term (18-day) survival in ferrets. We analyzed viral replication, type and severity of meningoencephalitis, infected cell types, and cellular responses to infection. We found viral replication to very high titers in ferret brain, closely correlating with severity of meningoencephalitis. Viral antigens were detected predominantly in neurons, correlating with inflammatory lesions, and less frequently in astrocytes and ependymal cells during active infection. Mononuclear cell infiltrates were observed in early stages predominantly in cerebral cortex, brainstem, and leptomeninges, and less commonly in cerebellum and other areas. Astrogliosis was mild at day 4 post-infection, but robust by day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced severity of brain injury, and promoted higher survival rates.

Functional Interferon System is Required for Clearance of Lassa Virus

Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever (LF) in humans, a deadly disease endemic to West Africa that results in 5,000 to 10,000 deaths annually. Here we present results demonstrating that functional type I and type II interferon (IFN) signaling is required for efficient control of LASV dissemination and clearance.

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