Daniel E. Hassett

Immune Responses following Neonatal DNA Vaccination Are Long-Lived, Abundant, and Qualitatively Similar to Those Induced by Conventional Immunization

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 animals 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.

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.

Direct Ex Vivo Kinetic and Phenotypic Analyses of CD8+ T-Cell Responses Induced by DNA Immunization

ABSTRACT 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.

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.

Coxsackievirus replication and the cell cycle: a potential regulatory mechanism for viral persistence/latency

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.

Peptide vaccination of mice immune to LCMV or vaccinia virus causes serious CD8+ T cell-mediated, TNF-dependent immunopathology

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.

Vaccinia Virus Infection during Murine Pregnancy: a New Pathogenesis Model for Vaccinia Fetalis

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.

Smallpox infections during pregnancy, lessons on pathogenesis from nonpregnant animal models of infection

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.