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J L Whitton

Publications and source records attributed to J L Whitton.

At least 73 records · Page 4Linked to original sources
PubMed1996

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. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.

Animals↗
PubMed1995

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. These CTL "escape" variants nevertheless elicited sufficient host-protective activity in vivo to abort acute infection and prevent the occurrence of persistent infection. This protection was CD8+ lymphocyte mediated and associated with the generation of a novel (for H-2b mice) CTL response to the viral L protein. Hence CTL epitopes form a hierarchy, in which responses to "weak" epitopes are suppressed in the presence of "stronger" epitopes. Mutation in the strong epitopes may be of limited biological significance since the host can mount a protective response directed against the second level (weak) epitopes.

Amino Acid Sequence↗
PubMed1995

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. These cells exhibited a marked reduction in their ability to support viral infection. Here we report the construction of RNA molecules that contain one or two antiviral ribozymes, each specific for a different cleavage site on the genome of the target virus, lymphocytic choriomeningitis virus (LCMV), and each contained in a self-cleavage cassette comprising cis-acting ribozymes designed to release the antiviral molecules from the transcript. In vitro studies showed that both antiviral ribozymes were released properly from the RNAs following cleavage by the flanking ribozymes and that these released ribozymes functioned as expected in cleaving the target virus RNA. These self-cleaving cassettes have been clones into a retroviral vector downstream of, but in the same transcript as, the chloramphenicol acetyltransferase (CAT) gene. Thus, we hoped to employ CAT as a surrogate marker of ribozyme transcription. Stably transformed cell lines were established. Cleavage by the cis-acting ribozymes was incomplete, as assessed by Northern blot analysis and by the ability of transformed cells to produce infectious retroviral particles. Nevertheless, the antiviral ribozyme sequences exerted effects in tissue culture. LCMV RNA levels in ribozyme-expressing cells were suppressed, and infectious virus yields were decreased by up to 95% compared with normal cells and with cells expressing inverted ribozymes. The antiviral effects correlated with CAT levels, but there was no significant difference between cell lines expressing a single ribozymes and those expressing two.

3T3 Cells↗
PubMed1995

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. CVB3 was able to infect all strains used (C57BL/6, CD4ko, and beta-microglobulin ko [beta 2Mko]), and following intraperitoneal injection, two disease processes could be distinguished. First, the virus caused early (3 to 7 days postinfection) death in a viral dose-dependent manner. Immunocompetent C57BL/6 mice were highly susceptible (50% lethal dose = 70 PFU), while immunodeficient transgenic ko mice were less susceptible, showing 10- and 180-fold increases in the 50% lethal dose (for CD4ko and beta 2Mko mice, respectively). Second, a histologic examination of surviving CD4ko mice at 7 days postinfection revealed severe myocarditis; the inflammatory infiltrate comprised 40 to 50% macrophages, 30 to 40% NK cells, and 10 to 20% CD8+ T lymphocytes. The infiltration resolved over the following 2 to 3 weeks, with resultant myocardial fibrosis. In vivo depletion of CD8+ T lymphocytes from these CD4ko mice led to a marked reduction in myocarditis and an increase in myocardial virus titers. beta 2Mko mice, which lack antiviral CD8+ T cells, are much less susceptible to early death and to the development of myocarditis. We conclude that our data support a strong immunopathologic component in CVB3-induced disease and implicate both CD4+ and CD8+ T cells. Compared with immunocompetent animals, (i) mice lacking CD4+ T cells (CD4ko) were more resistant to virus challenge, and (ii) mice lacking CD8+ T cells (beta 2Mko and in vivo-depleted CD4ko) showed enhanced survival and a reduced incidence of the later myocarditis. Nevertheless, the picture is complex, since (iii) removal of the CD4+ component, while protecting against early death, greatly magnified the severity of myocarditis, and (iv) removal of the CD8+ cells from CD4ko mice, although protecting against early death and later myocarditis, led to markedly increased virus titers in the heart. These data underscore the complex balance between the costs and benefits of effective antiviral immune responses.

Animals↗
PubMed1995

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. The protection conferred is incomplete, but it is most probably mediated by the induced cytotoxic T lymphocytes.

Animals↗
PubMed1994

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. The expression of cytokine mRNA (particularly IFN-gamma) in organs of LCMV-infected mice treated with anti-IFN-alpha/beta coincided with infiltration of lymphocytes and tissue destruction. Furthermore, a reduced number of infiltrating leukocytes in the brain and cervical lymph nodes and a low expression of cytokine mRNA in the brain was observed in anti-IFN-alpha/beta-treated animals. In total, the findings support the view that neutralization of IFN-alpha/beta leads to extensive LCMV replication in the viscera. The therapeutic effects of anti-IFN-alpha/beta antiserum seem to be independent of the functional capacity of T cells but probably result in a dispersion of activated T cells throughout the body of LCMV-infected mice. Absence of IFN-alpha/beta expression in the central nervous system is proposed as the mechanism behind the IFN-alpha/beta-dependent targeting of T cells to the brain.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

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. The majority of these LCMV-infected TCR-transgenic mice show a substantial meningitis of delayed onset, that resolves without causing any obvious clinical impairment. This inflammatory process depends on the involvement of V beta 8+ T cells, but does not require the participation of the CD4+ subset. The cytotoxic effectors that develop in both the transgenic mice and the CBA/Ca controls are lytic for target cells infected with a vaccinia construct expressing genes encoding the putative polymerase protein of LCMV. Limiting the available TCR repertoire to lymphocytes with a single V beta phenotype (not required for the generation of potent effectors in wild-type mice) thus modifies the development of the lethal neuropathology characteristic of LCMV infection, although the CD8+ cytotoxic T lymphocyte response is not greatly compromised.

Acute Disease↗
PubMed1994

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.

Animals↗
PubMed1993

Enhancement of autoimmune disease using recombinant vaccinia virus encoding myelin proteolipid protein.

Viral infections have been associated with the initiation and exacerbations often observed with autoimmune disease. Mechanisms by which viruses may play a role in the development of autoreactive immune responses include polyclonal activation of B and T cells, molecular mimicry, viral infection of immune cells, exposure of sequestered antigens, or altered host cell expression (neoantigen or altered self) in virus infected host cells. We have been studying the immune response generated to self proteins in association with viral infection. Here we evaluate the effects of viral infection on the development of an autoimmune disease of the central nervous system, experimental allergic encephalomyelitis. A vaccinia virus construct, VVplp was made containing the coding region for rat myelin proteolipid protein (PLP). Cells infected with VVplp were found to express PLP protein in vitro. Central nervous system disease was not detectable in mice vaccinated with VVplp. However, mice vaccinated with VVplp and later challenged with encephalitogenic peptides derived from PLP were found to have enhanced disease with earlier onset of symptoms when compared to mice treated with a control vaccinia virus construct. This enhancement of disease was found to peak at 10 days post challenge with the encephalitogenic PLP peptide. Clinical disease and an inflammatory response in the central nervous system was evident in mice previously vaccinated with VVplp but not in control vaccinated mice at this time. These results indicate that prior infection with a virus capable of coding for self protein can predispose the host to an accentuated autoimmune response.

Animals↗
PubMed1993

A "string-of-beads" vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge.

We have previously demonstrated that induction of antiviral cytotoxic T lymphocytes (CTL), in the absence of antiviral antibodies, can confer protection against a lethal-dose virus challenge. Here we extend those findings as follows. First, three discrete viral CTL epitopes expressed from minigenes encoding peptides as short as 12 amino acids can be recognized when expressed from recombinant vaccinia virus; second, concentrating on two of the three epitopes, we show that these vaccinia virus recombinants can confer protection in a major histocompatibility complex (MHC)-restricted manner; third, the minigenes can be fused to generate a "string of beads," and the close proximity of the two epitopes within one oligopeptide does not disrupt recognition of either epitope; fourth, this string-of-beads vaccine, in contrast to the single epitope vaccines, can protect on both MHC backgrounds; and, fifth, CTL to different epitopes may act synergistically, as protection is improved when the vaccine contains more than one CTL epitope for a given MHC background.

Amino Acid Sequence↗
PubMed1993

An anti-lymphocytic choriomeningitis virus ribozyme expressed in tissue culture cells diminishes viral RNA levels and leads to a reduction in infectious virus yield.

Ribozymes, RNA molecules which cleave RNA in a sequence-specific manner, are a promising tool in the development of specific antiviral therapies. The viruses most susceptible to ribozymes may be those in which all aspects of the viral life cycle depend on RNA, with no DNA intermediate. Consequently, we have chosen as a model one such virus, the arenavirus lymphocytic choriomeningitis virus (LCMV), and have previously reported the design of specific anti-LCMV ribozymes (Z. Xing and J. L. Whitton, J. Virol. 66:1361-1369, 1992). Here we describe the establishment of several cell lines, each stably expressing an antiarenaviral ribozyme of different specificity. Expression of a single ribozyme leads to a reduction in LCMV RNA levels, and stimulation of ribozyme transcription amplifies the effect. Target site selection may be an important determinant of antiviral effectiveness, since the extent of the antiviral effect, measured by assay of viral RNA, varies with the specificity of the antiviral ribozyme expressed. Furthermore, infectious virus production is reduced approximately 100-fold. This effect is LCMV specific, as yield of a related arenavirus is not similarly curtailed. We are currently investigating the mechanism underlying the ribozyme-mediated antiviral effect.

Animals↗
PubMed1993

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. The virus persists in many cell types, including lymphocytes and macrophages. We show that prior immunization with a vaccine designed to induce CTL (in the absence of antiviral antibody) confers complete protection against subsequent establishment of persistence in all tissues analyzed. The vaccine can be designed to express as few as 10 amino acids of a viral protein that comprise the CTL epitope. Further, two CTL epitopes for two discrete MHC haplotypes can be successfully used in a single vaccine that protects both strains of mice. Hence, a "string of CTL epitopes" (beads) concept for vaccination is feasible. Finally, the CTL vaccine provided protection against the establishment of persistence by an immunosuppressive virus.

Amino Acid Sequence↗
PubMed1993

A rapid and simple method for determining the DNA sequences of fragments inserted into vaccinia virus.

Recombinant virus vectors such as vaccinia virus, adenovirus and herpesvirus are frequently used to express a variety of foreign products. A rapid method allowing the precise identification of recombinants would be useful to confirm the nature of a newly produced recombinant and, in particular, to discriminate between recombinants bearing near-identical foreign products. Using vaccinia virus, we describe a method that in one day provides sequence analysis of the recombinant viral DNA.

Base Sequence↗
PubMed1992

A common antiviral cytotoxic T-lymphocyte epitope for diverse major histocompatibility complex haplotypes: implications for vaccination.

Of nine established murine haplotypes, mice of three types (H-2d, H-2u, and H-2q) possess major histocompatibility complex class I glycoproteins able to present an identical viral peptide for recognition and lysis by virus-specific cytotoxic T lymphocytes. Incorporation of this viral epitope into a recombinant vaccinia vaccine and administration of a single dose protects mice with these three haplotypes from an ordinarily lethal challenge of virus. Hence, a common epitope can exist. The sharing of the ability to bind such epitopes among different MHC haplotypes underscores the feasibility of developing an effective cytotoxic T-lymphocyte vaccine for outbred populations like humans.

Amino Acid Sequence↗
PubMed1992

LCMV-specific, class II-restricted cytotoxic T cells in beta 2-microglobulin-deficient mice.

Intracranial infection of normal mice with lymphocytic choriomeningitis virus (LCMV) causes meningitis and death mediated by CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs). beta 2-Microglobulin-deficient mice (beta 2M-/-) do not express functional MHC class I proteins and do not produce significant numbers of CD8+ T cells. When beta 2M-/- mice were infected with LCMV, many died from LCMV disease and produced a specific response to LCMV mediated by CD4+ CTLs that were class II-restricted. In these mice, CD4+ CTLs may compensate for the lack of CD8+ CTLs.

Animals↗