CD8 T cells play a key role in clearing primary virus infections and in protecting against subsequent challenge. The potent antiviral effects of these cells make them important components of vaccine-induced immunity and, because of this, peptide vaccines often contain epitopes designed to induce strong CD8 T cell responses. However, the same effector functions that protect the host also can be harmful if they are not tightly regulated, and virus-specific CD8 T cells are a frequent cause of immunopathology. Here, we report that the administration of peptide to virus-immune recipient mice can lead to the synchronous activation of preexisting virus-specific CD8 T cells with serious, and even lethal, consequences. Mice infected with LCMV or vaccinia virus developed rapid and profound hypothermia following injection of cognate synthetic peptides, and LCMV-infected mice frequently died within hours. Detailed analyses of the LCMV infected mice revealed enterocyte apoptosis and implicated TNF produced by peptide-specific CD8 T cells as the major mediator of disease. The caspase inhibitor zVADfmk had no demonstrable effect on the development of hypothermia, but diminished enterocyte apoptosis and greatly reduced the number of deaths. These findings, if similarly observed in patients, counsel caution when administering powerful immunogens such as peptide vaccines to individuals who may have a large preexisting pool of epitope-specific CD8 T cells.

Coxsackieviruses (CV) are characterized by their ability to cause cytopathic effects in tissue culture and by their capacity to initiate acute disease by inducing apoptosis within targeted organs in vivo. These viruses are considered highly cytolytic, but can establish persistence/latency in susceptible cells, indicating that a regulatory mechanism may exist to shut off viral protein synthesis and replication under certain situations. The persistence of coxsackieviral RNA is of particular medical interest due to its association with chronic human diseases such as dilated cardiomyopathy and chronic inflammatory myopathy. Here, we discuss the potential mechanisms regulating coxsackievirus replication, and the ability of viral RNA to remain in an apparent latent state within quiescent cells.

ABSTRACT Vaccinia fetalis, the vertical transfer of vaccinia virus from mother to fetus, is a relatively rare but often fatal complication of primary vaccinia virus vaccination during pregnancy. To date there has been no attempt to develop an animal model to study the pathogenesis of this acute viral infection in vivo. Here we report that infection of gestating BALB/c mice by either intravenous or intraperitoneal routes with the Western Reserve strain of vaccinia virus results in the rapid colonization of the placenta and vertical transfer of virus to the developing fetus. Systemic maternal infections during gestation lead to the death of all offspring prior to or very shortly after birth. Using in situ hybridization for vaccinia virus mRNA to identify infected cells, we show that the virus initially colonizes cells lining maternal lacunae within the trophospongium layer of the placenta. The study of this model will significantly enhance our understanding of the pathogenesis of fetal vaccinia virus infections and aid in the development of effective treatments designed to reduce the risk of vaccinia virus-associated complications during pregnancy.

Both vaccinated and unvaccinated women during pregnancy who contract variola virus, the causative agent of smallpox, suffer much higher mortality rates than nonpregnants. Furthermore, acute maternal smallpox leads to spontaneous abortion, premature termination of pregnancy and early postnatal infant mortality. The mechanisms governing the abortifacient activity of smallpox, as well as the enhanced susceptibility of gestating women to lethal disease, have remained largely unexamined. Experimental poxvirus infections in nonpregnant small animal models have revealed that T helper type 1 (TH1) cytokines promote efficient resolution of these infections whereas type 2 (TH2) cytokines enhance viral pathogenesis. These data, combined with recent understanding of how the immune system is modulated by pregnancy, may offer important clues as to the increased pathogenesis of variola in pregnant women. The aim of this review is to bring together the current literature on the effects of poxvirus infections in nonpregnant hosts, as well as the effects of pregnancy on the immune system, in order to develop unifying concepts that may provide insight into the pathogenesis of variola during pregnancy and why prior vaccination with vaccinia virus the live anti-variola vaccine offers less protection to pregnant women and their unborn children.

Neonates are particularly susceptible to coxsackievirus infections of the central nervous system (CNS), which can cause meningitis, encephalitis, and long-term neurological deficits. However, viral tropism and mechanism of spread in the CNS have not been examined. Here we investigate coxsackievirus B3 (CVB3) tropism and pathology in the CNS of neonatal mice, using a recombinant virus expressing the enhanced green fluorescent protein (eGFP). Newborn pups were extremely vulnerable to coxsackievirus CNS infection, and this susceptibility decreased dramatically by 7 days of age. Twenty-four hours after intracranial infection of newborn mice, viral genomic RNA and viral protein expression were detected in the choroid plexus, the olfactory bulb, and in cells bordering the cerebral ventricles. Many of the infected cells bore the anatomical characteristics of type B stem cells, which can give rise to neurons and astrocytes, and expressed the intermediate filament protein nestin, a marker for progenitor cells. As the infection progressed, viral protein was identified in the brain parenchyma, first in cells expressing neuron-specific class III beta-tubulin, an early marker of neuronal differentiation, and subsequently in cells expressing NeuN, a marker of mature neurons. At later time points, viral protein expression was restricted to neurons in specific regions of the brain, including the hippocampus, the entorhinal and temporal cortex, and the olfactory bulb. Extensive neuronal death was visible, and appeared to result from virus-induced apoptosis. We propose that the increased susceptibility of the neonatal CNS to CVB infection may be explained by the virus' targeting neonatal stem cells; and that CVB is carried into the brain parenchyma by developing neurons, which continue to migrate and differentiate despite the infection. On full maturation, some or all of the infected neurons undergo apoptosis, and the resulting neuronal loss can explain the longer-term clinical picture.

Neonates are thought to mount less vigorous adaptive immune responses than adults to antigens and infectious agents. This concept has led to a delay in the administration of many currently available vaccines until late infancy or early childhood. It has recently been shown that vaccines composed of plasmid DNA can induce both humoral and cell-mediated antimicrobial immunity when administered within hours of birth. In most of these studies, immune responses were measured weeks or months after the initial vaccination, and it is therefore questionable whether the observed responses were actually the result of priming of splenocytes within the neonatal period. Here we show that DNA vaccination at birth results in the rapid induction of antigen-specific CD8(+) T cells within neonatal life. Analyses of T-cell effector functions critical for the resolution of many viral infections revealed that neonatal and adult CD8(+) T cells produce similar arrays of cytokines. Furthermore, the avidities of neonatal and adult CD8(+) T cells for peptide and the rapidity with which they upregulate cytokine production after recall encounters with antigen are similar. Protective immunity against the arenavirus lymphocytic choriomeningitis virus, which is mediated by CD8(+) cytotoxic T cells, is also rapidly acquired within the neonatal period. Collectively these data imply that, at least in the case of CD8(+) T cells, neonates are not as immunodeficient as previously supposed and that DNA vaccines may be an effective and safe means of providing critical cell-mediated antiviral immunity extremely early in life.

ABSTRACT Virus infections are devastating to neonates, and the induction of active antiviral immunity in this age group is an important goal. Here, we show that a single neonatal DNA vaccination induces cellular and humoral immune responses which are maintained for a significant part of the animal's life span. We employ a sensitive technique which permits the first demonstration and quantitation, directly ex vivo, of virus-specific CD8 + T cells induced by DNA immunization. One year postvaccination, antigen-specific CD8 + T cells were readily detectable and constituted 0.5 to 1% of all CD8 + T cells. By several criteria—including cytokine production, perforin content, development of lytic ability, and protective capacity—DNA vaccine-induced CD8 + memory T cells were indistinguishable from memory cells induced by immunization with a conventional (live-virus) vaccine. Analyses of long-term humoral immune responses revealed that, in contrast to the strong immunoglobulin G2a (IgG2a) skewing of the humoral response seen after conventional vaccination, IgG1 and IgG2a levels were similar in DNA-vaccinated neonatal and adult animals, indicating a balanced T helper response. Collectively, these results show that a single DNA vaccination within hours or days of birth can induce long-lasting CD8 + T- and B-cell responses; there is no need for secondary immunization (boosting). Furthermore, the observed immune responses induced in neonates and in adults are indistinguishable by several criteria, including protection against virus challenge.

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.

ABSTRACTDNA immunization induces antibodies to the encoded protein, which indicates that the protein must gain access to the extracellular milieu, allowing it to interact with naı̈ve 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.

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 naive 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 CD81 T cells. In addition, there was no skewing of the immunoglobulin G isotypes in the absence of perforin. DNA immunization is unique in that antigenic proteins are synthesized within the transfected cell in the absence of any associated infectious agent, and the vaccine itself contains no soluble protein that could initiate humoral immune responses. Thus, if the immunizing plasmid expresses a cytoplasmic pro- tein which is relatively noncytopathic and is unable to be ef- fectively processed endogenously by the major histocompati- bility complex (MHC) class II antigen presentation pathway, then it might be ineffective in inducing humoral immune re- sponses. The nucleoprotein (NP) of lymphocytic choriomenin- gitis virus (LCMV) meets these criteria, yet intramuscular in- jection with a plasmid expressing the NP (pCMV-NP) has been shown by us and others to induce antibody responses in mice (13, 23, 24). Antibodies are also induced following DNA im- munization with plasmids expressing other cytoplasmic anti- gens, such as the measles virus and influenza virus NPs (4, 18, 21). How might such antigens be released from the transfected cell? We hypothesized that the development of CD81 antigen- specific cytotoxic T lymphocytes (CTL) could lead to the rec- ognition and lysis of cells expressing plasmid-derived NP, re- sulting in the liberation of protein into the extracellular milieu, where it can then interact with B cells and antigen-presenting cells (APCs). CTL-mediated release of LCMV NP occurs dur- ing virus infection (12), and the possibility that it occurs following DNA immunization was strengthened by our observations of a profound myositis following intramuscular inoculation of pCMV-NP into LCMV-infected or -immune mice; the peak of the inflammatory infiltrate in acutely infected mice coincided with the development of anti-LCMV CTL, and destruction of muscle cells occurred (8, 22). Professional APCs, which are known to be a source of plasmid-expressed antigens (5) and which appear to be the cell type responsible for initiating immune responses following DNA immunization (6, 7), can also be recognized and lysed by CTL. Dendritic cells infected with human immunodeficiency virus are susceptible to lysis in vitro by CTL (11), and there is evidence to suggest that CD81

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.

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 γ (IFNγ) by cells of the innate immune system. Although IFNγ induction by CpG elements within plasmid DNA has been documented in vitro and more recently in vivo, and coimmunization with plasmids expressing IFNγ has been shown to enhance DNA-immunization-induced immune responses, it is unclear if IFNγ is necessary for successful DNA immunization. To address this issue, we compared humoral and cellular immune responses in wild-type and IFNγ-deficient mice vaccinated with a plasmid (pCMVNP) expressing the nucleoprotein gene from the arenavirus lymphocytic choriomeningitis virus (LCMV). IFNγ-positive (BALB/c) and IFNγ-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 vaccinee. DNA vaccines also induced virus-specific cytotoxic T lymphocytes (CTL), even in the absence of IFNγ. 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, IFNγ 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 IFNγ 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 IFNγ 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, IFNγ is necessary for the development of IgG2a antibodies that may be crucial for protection against some pathogens.

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.

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 does-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.

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.

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. USA91, 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 vitroassays 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.

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.