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.

A multivalent minigene vaccine, containing B-cell, cytotoxic T-lymphocyte, and Th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen.

The development of safe and effective vaccines remains a major goal in the prevention, and perhaps treatment, of infectious diseases. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of the immune response. We originally demonstrated that two virus-specific cytotoxic T-lymphocyte (CTL) epitopes, from the same virus but presented by different major histocompatibility complex alleles, when linked in tandem as minigenes in a recombinant vaccinia virus, could confer complete protection against subsequent viral challenge.

Use of a nonviral vector to express a chimeric tRNA-ribozyme against lymphocytic choriomeningitis virus: cytoplasmic accumulation of a catalytically competent transcript but minimal antiviral effect.

RNA polymerase III promoters direct the ubiquitous, high-level, expression of small, stable RNAs such as tRNAs, and thus are attractive candidates for achieving stable expression of small therapeutic (e.g., antiviral) molecules, such as ribozymes or antisense RNAs.

CNS-Specific Ribozyme Expression.

Targeted genetic deletion is a powerful tool for analysis of gene function, but the standard approaches carry certain inescapable disadvantages. First, deletion is ubiquitous; tissue-specific knockout cannot be obtained. Second, temporal regulation of depletion is unattainable; the deleted functions are absent throughout the animal's development.

Vaccination protects beta 2 microglobulin deficient mice from immune mediated mortality but not from persisting viral infection.

Intracranial (i.c.) infection of immunocompetent mice with lymphocytic choriomeningitis virus (LCMV) results in immunopathological lethal meningitis mediated by CD8+ cytotoxic T lymphocytes (CTL).

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.

DNA immunization: effects of vehicle and route of administration on the induction of protective antiviral immunity.

The effectiveness of DNA immunization has been demonstrated in several model systems, usually following intramuscular injection of DNA in saline, or topical administration to the skin. In this study we have compared DNA delivered by three routes (intramuscular, intravenous, and intraperitoneal) and, for each route, in two vehicles (cationic liposome complex and pH sensitive liposome). These two lipid vehicles were evaluated because they are frequently used in gene therapy studies, but their immunogenicity has not been extensively studied.

A recombinant minigene vaccine containing a nonameric cytotoxic-T-lymphocyte epitope confers limited protection against Listeria monocytogenes infection.

We have previously shown that vaccines expressing virus-derived cytotoxic-T-lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice.

Virus encoding an encephalitogenic peptide protects mice from experimental allergic encephalomyelitis.

The association of viral infections with autoimmune central nervous system (CNS) diseases such as post-infectious encephalomyelitis and possibly multiple sclerosis (MS) prompted the investigation to understand how virus infection could modulate autoimmune responses. Recombinant vaccinia viruses encoding an encephalitogenic portion of myelin basic protein (MBP) were evaluated in an animal model for human demyelinating disease, experimental allergic encephalomyelitis (EAE). We have determined that mice vaccinated with recombinant viruses encoding an encephalitogenic region of MBP were protected from EAE.

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL.

The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis.

Coxsackievirus infections have previously been shown to cause acute or chronic myocarditis in humans, and several mouse models have been established to study the pathology of this disease. Myocardial injury may result from direct viral effects and/or may be immune mediated. To determine the relative roles of these processes in pathogenesis, we have compared coxsackievirus B3 (CVB3) infections of normal and immuno-compromised transgenic knockout (ko) mice.

CTL escape viral variants. II. Biologic activity in vivo.

The proteins of lymphocytic choriomeningitis virus (LCMV) contain only three known peptide regions that are processed and then held in place by the MHC class I H-2b (Db) glycoprotein on the cell's surface for recognition by LCMV-specific Db-restricted cytotoxic T lymphocytes (CTL). These peptides are from the glycoprotein (GP), amino acids 33-41 KAVYNFATC (GP1) and 276-286 SGVENPGGYCL (GP2), and the nucleoprotein (NP), 396-404. We have used CTL clones that recognized only GP1, GP2, and NP to select viral variants that upon infecting cells bearing H-2b molecules escaped recognition by virus-specific CTL directed against the viral GP (GP1 + GP2) mutant, termed GPV, or the viral GP and NP (GP1 + GP2 + NP) mutant, termed GPV+NPV.

Second malignant neoplasms following treatment for Wilm's tumor: a report from the National Wilms' Tumor Study Group.

Purpose: The study was undertaken to determine the incidence of second malignant neoplasms (SMNs) in patients treated for Wilms' tumor and demonstrate how the incidence varied with the initial treatment protocol.

Patients And Methods: Between October 1969 and December 1991, 5,278 assessable patients were enrolled onto the National Wilms' Tumor Study (NWTS) and by the end of 1993 had contributed 39,461 person-years to a follow-up study. Expected numbers of second cancers were calculated by applying national incidence rates to person-years classified by age, sex, and calendar year.

DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection.

DNA vaccination has been evaluated with the lymphocytic choriomeningitis virus (LCMV) model system. Plasmid DNA encoding the LCMV nucleoprotein, when injected intramuscularly, induces both antiviral antibodies and cytotoxic T lymphocytes. Injection of DNA encoding the nucleoprotein or the viral glycoprotein confers protection against normally lethal LCMV challenge in a major histocompatibility complex-dependent manner.

Antiviral activity of RNA molecules containing self-releasing ribozymes targeted to lymphocytic choriomeningitis virus.

Ribozymes catalytically cleave substrate RNA molecules in a sequence-specific manner. Engineered ribozymes can be developed and introduced into tissue culture cells to regulate gene expression and to inhibit viral replication. We have previously reported on the construction of cell lines that constitutively express a single antiviral ribozyme embedded in a lengthy RNA transcript.

Altered tissue distribution of viral replication and T cell spreading is pivotal in the protection against fatal lymphocytic choriomeningitis in mice after neutralization of IFN-alpha/beta.

IFN-alpha/beta have been shown to play a central role in the development of lymphocytic choriomeningitis and increasing attention has been focused on this group of cytokines as early regulatory factors directing T lymphocyte responses. In the present study, injection of antiserum to IFN-alpha/beta prevented the development of lymphocytic choriomeningitis, was associated with the absence of detectable expression of early 2'-5' oligo-adenylate synthetase mRNA and coincided with viremia of lymphocytic choriomeningitis virus (LCMV) followed by establishment of a persistent infection. The LCMV-specific cytotoxic T lymphocyte response was unchanged in cervical lymph nodes but decreased in the spleen of anti-IFN-alpha/beta-treated animals.

Limiting the available T cell receptor repertoire modifies acute lymphocytic choriomeningitis virus-induced immunopathology.

The invariably fatal immunopathological disease that follows intracerebral injection of CBA/Ca (H-2k) mice with 1000 PFU of lymphocytic choriomeningitis virus (LCMV) generally fails to develop in congenic mice transgenic for a V beta 8.1D beta 2J beta 2.3C beta 2 T cell receptor (TCR) gene.

Antisense treatment of viral infection.

In this chapter I have attempted to outline the rationale that underlies the antisense approach to treatment of virus infection, to catalog the effector molecules that are currently available, and to estimate the relative worth of each. In so doing I have tried to describe the criteria that might be employed in their design and the factors that may determine their efficacy in tissue culture and, perhaps, in vivo. Finally, I have described the few examples presently available that indicate that antisense approaches may one day be therapeutically useful in treatment of disease of viral or nonviral origin.

Vaccination to prevent persistent viral infection.

Persistent virus infections are increasingly being recognized as a significant cause of human morbidity and mortality. To establish persistence, a virus must establish infection and evade eradication by the host immune response, in particular by cytotoxic T lymphocytes (CTL). We have studied a virus that establishes persistence in part by suppressing the CTL response of the infected host.