Biomedical subjects

D E Hassett

Publications and source records attributed to D E Hassett.

11 recordsLinked to original sources

PubMed1999

Plasmid DNA vaccines are effective in the absence of IFNgamma.

Intramuscular injection of bacterially derived plasmid DNA results in the development of both humoral and cellular immune responses against plasmid-encoded antigens. Immunostimulatory CpG sequences within bacterial DNA are thought to enhance this process by stimulating the secretion of proinflammatory cytokines such as interferon gamma (IFNgamma) by cells of the innate immune system. Although IFNgamma induction by CpG elements within plasmid DNA has been documented in vitro and more recently in vivo, and coimmunization with plasmids expressing IFNgamma has been shown to enhance DNA-immunization-induced immune responses, it is unclear if IFNgamma is necessary for successful DNA immunization. To address this issue, we compared humoral and cellular immune responses in wild-type and IFNgamma-deficient mice vaccinated with a plasmid (pCMVNP) expressing the nucleoprotein gene from the arenavirus lymphocytic choriomeningitis virus (LCMV). IFNgamma-positive (BALB/c) and IFNgamma-negative (GKO) mice responded to DNA vaccination by the development of antigen-specific CD8(+) T cells, which were detectable directly ex vivo by intracellular cytokine staining and comprised 0.7-2.5% of all CD8(+) T cells in the vaccine. DNA vaccines also induced virus-specific cytotoxic T lymphocytes (CTL), even in the absence of IFNgamma. DNA vaccination of both mouse strains also was associated with a significant reduction in viral titers after LCMV challenge, indicating that, at least in the presence of other immune effector mechanisms, IFNgamma is not required for induction of protective anti-viral immunity by DNA immunization. No quantitative differences were observed in antiviral IgG levels among GKO and BALB/c vaccinees, although GKO mice did exhibit a significant reduction of the IgG2a:IgG1 ratio, in agreement with the previously documented requirement for IFNgamma in isotype switching to IgG2a. Immunized BALB/c mice produced similar levels of both IgG1 and IgG2a, indicating a mixed Th1/Th2 response to intramuscular immunization with pCMVNP. These results show that IFNgamma induction by bacterially derived plasmid DNA does not contribute to the magnitude of the antibody response and is not required for the induction or short-term maintenance of DNA-induced CTL. However, IFNgamma is necessary for the development of IgG2a antibodies that may be crucial for protection against some pathogens.

Animals↗

PubMed1999

DNA immunization: mechanistic studies.

DNA immunization works, as has been amply demonstrated in a variety of microbial and tumor models. However, the mechanisms which underpin its success remain unclear. Using intramuscular delivery of DNA, we wish to precisely define how DNA-encoded antigens induce CD8+ T-cells (most cytotoxic T-cells; CTL), CD4+ T-cells (mostly helper cells) and antibodies; and to use the accrued knowledge to rationally manipulate DNA vaccines, thus enabling us to optimize each of the above three types of immune response. We consider it likely that different mechanisms operate in each case. We have designed a DNA vaccine which induces CTL, but not antibodies. We will present evidence that CTL are induced by endogenously-synthesized protein, not by protein released from cells; and that in the absence of release of intact protein, antibodies are not induced, while CTL induction remains strong. We have used plasmid-encoded minigenes and have found that these short sequences also induce CTL; this, too, argues that CTL are induced by antigens presented following endogenous synthesis. We are attempting to determine how antigens are released from transfected cells, to interact with B-cells and induce antibodies, and are currently evaluating the CD4 responses induced by DNA vaccines.

Animals↗

PubMed1999

Induction of antiviral antibodies by DNA immunization requires neither perforin-mediated nor CD8(+)-T-cell-mediated lysis of antigen-expressing cells.

DNA immunization induces antibodies to the encoded protein, which indicates that the protein must gain access to the extracellular milieu, allowing it to interact with naïve B lymphocytes. It has been suggested that antigen release may be effected by cytotoxic-T-lymphocyte-mediated lysis of transfected antigen-expressing cells; this might be particularly important for the induction of responses to a noncytopathic, cytosolic protein. Here we show that the induction of antibody responses to one such DNA-encoded protein required neither perforin nor CD8(+) T cells. In addition, there was no skewing of the immunoglobulin G isotypes in the absence of perforin.

Animals↗

PubMed1998

Transgenic expression of IFN-alpha in the central nervous system of mice protects against lethal neurotropic viral infection but induces inflammation and neurodegeneration.

Type I IFNs, which include IFN-alpha, appear to have complex and broad-ranging actions in the central nervous system (CNS) that may result in protection or injury. To better understand these issues, we generated transgenic mice that produce IFN-alpha1 chronically from astrocytes. These glial fibrillary acidic protein-IFN-alpha transgenic mice developed a progressive inflammatory encephalopathy, with marked calcium mineralization, meninoencephalitis, gliosis, and neurodegeneration. Many features of this murine encephalopathy resembled those found in certain human encephalopathies of unknown etiology; these diseases, exemplified by Aicardi-Goutières syndrome and some viral encephalopathies, show increased intrathecal production of IFN-alpha. Our data suggest that IFN-alpha overproduction may be the primary factor initiating these human diseases. Following intracerebral infection with lymphocytic choriomeningitis virus, glial fibrillary acidic protein-IFN-alpha mice had significantly increased survival rates associated with markedly reduced virus titers and immune pathology in the brain but normal peripheral CTL responses. Therefore, the production of IFN-alpha in the CNS can be a two-edged sword that on the one hand confers protection against a lethal viral infection but on the other causes significant injury to the brain. These transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I IFNs in the intact CNS.

Animals↗

PubMed1997

Analysis of a temperature-sensitive vaccinia virus mutant in the viral mRNA capping enzyme isolated by clustered charge-to-alanine mutagenesis and transient dominant selection.

We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994) Proc. Natl. Acad. Sci. USA 91, 4554-4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis. In vitro assays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.

Alanine↗

PubMed1997

DNA immunization can stimulate florid local inflammation, and the antiviral immunity induced varies depending on injection site.

DNA immunization is being considered to augment, or even to supplant, more traditional methods of antiviral immunization. Different routes of administration lead to markedly different levels of marker protein expression, but only limited data are available concerning the antiviral responses induced by DNA inoculated by different routes, and their protective efficacy. In this report we evaluate antiviral immunity induced by inoculation of DNA by the intramuscular (i.m.) and intradermal (i.d.) routes, and make three novel observations. First, i.d. immunization is dose-dependent and, although not uniformly successful, can induce very high levels of cytotoxic T lymphocyte (CTL) activity, varying dependent on the vehicle in which the DNA is administered. Second, while antiviral immunity induced by i.m. DNA injection has been demonstrated by many groups, we show herein a marked difference in immunity depending on the muscle injected. Immunity induced by DNA injection of the anterior tibial muscle significantly exceeds that induced following injection of the quadriceps muscle as judged by three criteria, namely CTL induction, decrease in virus titer following nonlethal challenge, and survival following a normally lethal challenge dose of virus. Thirdly, we evaluate the local immune response induced following immunization with DNA encoding a viral antigen. We show that, when recipients are already immune to the encoded protein, a severe but localized inflammatory response may result.

Animals↗

PubMed1997

Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge.

Conventional vaccines are remarkably effective in adults but are much less successful in the very young, who are less able to initiate a mature immune response and who may carry maternal antibodies which inactivate standard vaccines. We set out to determine whether DNA immunization might circumvent these problems. We have previously shown that intramuscular injection of plasmid DNA encoding the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) is capable of inducing immune responses and protecting 50% of adult mice against lethal and sublethal challenge with LCMV. Here we demonstrate that mouse pups injected with the same plasmid hours or days after birth produce major histocompatibility complex-restricted, NP-specific cytotoxic T lymphocytes (CTL) that persist into adulthood; 48% of vaccinated pups responded to subsequent sublethal viral challenge by the accelerated production of anti-NP LCMV-specific CTL, indicating that these animals had been successfully immunized by the plasmid DNA. In addition, these mice showed a >95% reduction in splenic viral titers 4 days postinfection compared to control mice, demonstrating a more rapid control of infection in vivo. Furthermore, pups born of and suckled on LCMV-immune dams (and therefore containing passively acquired anti-LCMV antibodies at the time of DNA inoculation) responded to the DNA vaccine in a similar manner, showing that maternally derived anti-LCMV antibodies do not significantly inhibit the generation of protective immune responses following DNA vaccination. These findings suggest that, at least in this model system, DNA immunization circumvents many of the problems associated with neonatal immunization.

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PubMed1996

DNA immunization.

Inoculation with plasmid DNA vectors encoding immunogenic proteins induces both humoral and cell-mediated immune responses, which often provide protective immunity. Although many questions regarding the mechanism, efficacy and safety of DNA immunization remain to be addressed, this approach may offer a safer and more cost-effective alternative to conventional vaccines.

Animals↗

PubMed1994

Targeted construction of temperature-sensitive mutations in vaccinia virus by replacing clustered charged residues with alanine.

The feasibility of using "clustered charge-to-alanine" mutagenesis (replacement by alanine of two or more charged residues clustered in a five- or six-amino acid sequence) to create temperature-sensitive, conditionally lethal mutations in vaccinia virus was examined by creating nine mutations in the vaccinia virus gene G2R. G2R was chosen for this analysis because mutations in this gene confer selectable phenotypes. Specifically, vaccinia viruses that contain a wild-type copy of G2R nare sensitive the effects of the anti-poxvirus drug isatin-beta-thiosemicarbazone (IBT), while mutations in G2R that completely abolish the function of the G2R protein product confer dependence upon IBT for growth. A previously isolated mutant carrying a temperature-sensitive mutation that maps to G2R (Cts56) is resistant to IBT at the permissive temperature and dependent upon IBT at the restrictive temperature. Nine clustered charge-to-alanine mutants were examined. Four of the these mutants (AS1, AS4, AS6, and AS9) display some degree of temperature sensitivity in the function of the G2R gene product. AS1 is temperature sensitive for growth in both a plaque assay and in a one-step growth experiment. AS6 and AS9 form small plaques at the nonpermissive temperature and are temperature sensitive for growth in a one-step growth experiment. AS4 manifests its temperature sensitivity as temperature-dependent IBT resistance. Five of the mutations (AS2, AS3, AS5, AS7, and AS8) appeared to confer phenotypes indistinguishable from that of wild-type vaccinia. These results demonstrate that temperature-sensitive conditionally lethal mutants can be created in vaccinia virus by altering the charge characteristics of essential viral proteins.

Alanine↗

PubMed1992

Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone.

Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue plasminogen activator (tPA), plays a crucial role in the regulation of fibrinolysis. Both hepatocytes and endothelial cells have been implicated as major sources of plasma PAI-1. To study the relative contribution of these cell types to hepatic PAI-1 production, we have separated hepatocytes and hepatic sinusoidal endothelial cells by fractionation of freshly isolated rat livers using metrizamide density gradients and centrifugal elutriation. In untreated animals, PAI-1 messenger RNA (mRNA) was detected only in the purified endothelial cell fraction, and not in the hepatocyte fraction or in unfractionated liver. However, when the animals were treated with dexamethasone, PAI-1 mRNA expression was transiently induced in the liver. This induction paralleled the appearance of PAI-1 mRNA in purified hepatocytes, while PAI-1 expression in sinusoidal endothelial cells was unchanged. Four hours after dexamethasone treatment, plasma PAI-1 levels were increased approximately twofold over levels measured in animals treated with the diluent alone. These data suggest that PAI-1 production by hepatocytes may contribute to elevated plasma PAI-1 levels in the setting of acute injury and stress.

Animals↗

PubMed1990

Genetic analysis of suppressors of the veA1 mutation in Aspergillus nidulans.

Light-dependent conidiation in the filamentous ascomycete, Aspergillus nidulans, is contingent on the allelic state of the velvet (veA) gene. Light dependence is abolished by a mutation in this gene (veA1), which allows conidiation to occur in the absence of light. We have isolated and characterized six extragenic suppressors of veA1 that restore the light-dependent conidiation phenotype. Alleles of four genes, defined by complementation tests, were subjected to extensive genetic and phenotypic analysis. The results of light-dark shifting experiments and the phenotypes of double mutant combinations are consistent with the possibility that the expression of the light-dependent phenotype is regulated by specific interactions of the suppressor gene products with the velvet gene product and with each other.

Alleles↗