DNA Immunization with Minigenes: Low Frequency of Memory Cytotoxic T Lymphocytes and Inefficient Antiviral Protection Are Rectified by Ubiquitination

Abstract

Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in ∼40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses—the presence of lytic activity following secondary stimulation—does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.

Incomplete effector/memory differentiation of antigen‐primed CD8+ T cells in gene gun DNA‐vaccinated mice

“…Several groups have tried to use DNA constructs that encode only a short immunodominant peptide from a given pathogen [10][11][12]. Such vector constructs are often referred to as minigenes and will in most circumstances lead to priming of only a limited lymphocyte sub-population, depending on MHC restriction.…”

“…Such vector constructs are often referred to as minigenes and will in most circumstances lead to priming of only a limited lymphocyte sub-population, depending on MHC restriction. Vaccination with minigenes alone, however, has been found to be inefficient and to produce short-lived protection [12][13][14]. Various strategies to improve the minigene approach have therefore been developed.…”

New technologies for vaccine development

“…Computational models for identification of B-cell epitopes have been reported [Van Regenmortel and Pellequer, 1994;Van Regenmortel, 1996], but these are still not very reliable. Vaccines based on immune epitopes have been reported to induce protection in animal models [Donnelly et al, 1995;Rodriguez et al, 1998;Sette and Sidney, 1999]. The development of these vaccines targets potential advantages over conventional vaccines -improved efficiency, better control of immunization regimens, and improved safety [Ishioka et al, 1999].…”

DNA immunization and central nervous system viral infection

“…Third, the space limitation of most potential viral vectors does not apply to DNA vaccines, since many different plasmids could be contained in a single vaccine "cocktail." Fourth, it is possible to manipulate the immune response induced--for example, by directing plasmid-encoded proteins to selectively induce CD8 ÷ T cells (Rodriguez et al, 1997;Rodriguez et al, 1998)--or to enhance induction of CD4 ÷ T cells (Rodriguez and Whitton, unpublished data). Fifth, DNA vaccines should be safe, and easy to produce cheaply, in quantity, and at a high level of purity.…”

“…The LCMV model has allowed the demonstration of the exquisite flexibility of DNA vaccines. If the LCMV NP gene is fused to the host protein ubiquitin, the resulting protein is targeted for very rapid intracellular degradation; a plasmid encoding this ubiquitin-NP fusion induces enhanced protection against intracranial challenge (Rodriguez et al, 1997), perhaps because it increases the precursor frequency of NP-specific CTLs (Rodriguez et al, 1998). In addition, the LCMV model has been used to study neonatal DNA immunization.…”