Quantitative and qualitative analyses of the immune responses induced by a multivalent minigene DNA vaccine

Abstract

Vaccines containing minigenes — isolated antigenic epitopes encoded by short open reading frames — can, under certain circumstances, confer protective immunity upon the vaccinee. Here we evaluate the efficacy of the minigene vaccine approach using DNA immunization and find that, to be immunogenic, a minigene-encoded epitope requires a perfect “Kozak” translational initiation region. In addition, using intracellular cytokine staining, we show that immunization with a plasmid encoding a full-length protein induces epitope-specific CD8⁺ T cells which are detectable directly ex vivo, and constitute ∼2% of the vaccinee’s splenic CD8⁺ T cells. In contrast, such cells are undetectable directly ex vivo in recipients of a minigene vaccine. Nevertheless, the minigene plasmid does induce a low number of epitope-specific CD8⁺ T cells, which can be amplified to detectable levels by in vivo stimulation. Indeed, 4 days after in vivo stimulation (by virus infection), all vaccinated mice — regardless of whether they had been vaccinated with the minigene or with the full-length gene — had similar numbers of epitope-specific CD8⁺ T cells. However, despite these strong responses at 4 days post-infection, recipients of the minigene vaccine showed no enhanced ability to limit virus replication and dissemination. We therefore observe a dichotomy; minigene vaccinees are not protected, despite the presence of strong virus-specific immune responses at 4 days post-challenge. We suggest that the protective benefits of vaccination exert themselves very soon — perhaps within minutes or hours — after virus challenge. If the vaccine-induced immune response is too low to achieve this early protective effect, virus-specific T cells will expand rapidly, but ineffectually, leading to the strong but non-protective response measured at 4 days post-infection. Thus, vaccine-induced immunity should be monitored very early in infection, since the extent to which these responses may later be amplified is largely irrelevant to the protection observed.

Introduction

Conventional vaccines have contributed enormously to human health worldwide, yet infections remain leading causes of death in many countries. Significant efforts are being made to develop new and effective vaccine strategies. For example, injection of plasmid DNA was shown several years ago to induce immune responses against the encoded genes [1], and these responses could protect against subsequent viral challenge [2]. DNA immunization has subsequently been shown to work in a large number of model systems, and human trials are under way; the comparative advantages and disadvantages of conventional and DNA vaccines have been reviewed [3]. In another novel approach to vaccination, we [4] and others [5] showed that isolated epitopes, for which we coined the term “minigenes”, could be used to confer protection against virus challenge, and that cytotoxic T lymphocyte (CTL) epitopes presented by different MHC class I alleles could be combined in a “string of beads” to protect several haplotypes [6]. These studies have since been confirmed and extended, and it is now clear that at least 10 CTL epitopes can be linked in a multivalent minigene vaccine [7]. The minigene approach is not limited to CTL epitopes; we [8] and others [9] have shown that T-helper and antibody responses can also be induced by appropriate minigene sequences. More recently, these two techniques have been combined, with the evaluation of DNA vaccines encoding minigenes; this has recently been reviewed [10].

In the present study we quantitate the immune responses induced by a minigene DNA vaccine, and compare it to the response induced by a vaccine encoding a full-length protein. In general, the cellular immune responses induced by DNA vaccines have not been detectable directly ex vivo; instead, some form of antigen-specific restimulation — for example, in vivo virus infection, or in vitro restimulation with peptide-coated cells — has been required to amplify the responses to detectable levels. However the technique of intracellular cytokine staining permits the direct ex vivo detection of low numbers of antigen-specific cells, and here we use this to directly enumerate, for the first time, the CD8⁺ T cell responses induced by minigene and full-length DNA vaccines. In addition, we characterize the protective efficacy of minigene DNA vaccination, and show that the most important correlate of protective immunity appears to be the host’s ability to respond immediately to the virus challenge. Finally we demonstrate, at least for a minigene vaccine, the importance of the “Kozak” sequences which flank the translational initiation codon.