Enhancement of protein vaccine potency by in vivo electroporation mediated intramuscular injection

Abstract

Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for innovative approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the employment of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses. Another approach involves employing a method capable of improving the delivery of protein-based vaccine intramuscularly to lead to the slow release of the protein, resulting in improved vaccine potency. In the current study, we aimed to determine whether intramuscular injection of protein-based vaccines in conjunction with CpG followed by electroporation can lead to increased delivery of the protein-based vaccine into muscle cells, resulting in enhanced protein-based vaccine potency. We found that intramuscular injection followed by electroporation can effectively transduce the protein-based vaccine into the muscle cells. Furthermore, we found that intramuscular vaccination with OVA protein in combination with CpG followed by electroporation generates the best OVA-specific CD8+ T cell immune responses as well as the best protective and therapeutic antitumor effects in vaccinated mice. CD8+ T cells were found to play an important role in the observed protective antitumor effects generated by the vaccination. Similar results were observed using the HPV-16 E7 protein-based vaccination system. Thus, our data indicate that intramuscular administration of protein-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+ T cell immune responses. The clinical implications of the study are discussed.

Introduction

Antigen-specific immunotherapy using protein-based vaccines has emerged as a potentially promising approach for the generation of antigen-specific immune responses. In general, protein-based vaccines are safe and have the ability to bypass MHC restriction (for reviews see [1], [2]). Various protein-based vaccines have been tested in clinical trials and found to be safe and well tolerated [3], [4], [5]. However, they are weakly immunogenic and usually induce low level of CTL responses [6], [7]. Consequently, most of the research in this area has focused on the identification of innovative adjuvants to enhance protein-based vaccine potency in generating antigen-specific CD8+ T cells (for review, see [8]).

One approach to enhance protein-based vaccine potency is the employment of “danger” signals such as toll-like receptor (TLR) ligands that stimulate dendritic cells to mature and differentiate into potent activators of antigen-specific T cells (for review, see [9]). It is clear that TLRs play a crucial role in enhancing innate and adaptive immune responses (for reviews, see [10], [11], [12], [13]). TLR ligands have emerged as a promising new class of vaccine adjuvants, particularly the oligodeoxynucleotides containing one or more unmethylated CpG dinucleotides (CpG-ODNs), which target TLR9 [14]. It has been shown that CpG-ODNs can stimulate innate immunity and confer protection against a variety of bacterial and viral infections [15], [16]. CpG-ODNs have previously been used in combination with protein-based vaccines to improve vaccine potency in several studies [17], [18], [19]. In fact, CpG ODNs have been employed in combination with HPV-16 E7 long peptide-based vaccines to generate significant antitumor effects against E7-expressing tumors [20].

Another strategy for increasing protein-based vaccine potency is to identify methods for retaining the protein for slow release into the immune system. We reasoned that a method capable of improving the delivery of protein-based vaccine intramuscularly may potentially lead to the slow release of the protein, resulting in improved vaccine potency. In the current study, we have employed electroporation to improve the delivery of protein-based vaccines into muscle cells. Electroporation has been used to improve transfection efficiency of DNA into muscle cells to improve DNA vaccine potency [21], [22], [23]. Electroporation involves the administration of the vaccine into the muscle by needle injection, followed by a brief electrical pulse to the surrounding tissue. A transient increase in the permeability of the plasma membrane induced by the electrical current allows increased uptake of the DNA plasmid [24], [25]. Thus, the technology may potentially be used to improve the uptake of protein-based vaccines by muscle cells.

In the current study, we plan to determine whether intramuscular injection of protein-based vaccines in conjunction with CpG followed by electroporation can lead to increased delivery of the protein-based vaccine into muscle cells to enhance protein-based vaccine potency. We found that intramuscular injection followed by electroporation can most effectively transduce the protein-based vaccine into the muscle cells. Furthermore, we found that intramuscular vaccination with OVA protein in combination with CpG followed by electroporation generates the best OVA-specific CD8+ T cell immune responses as well as the best protective and therapeutic antitumor effects in vaccinated mice. CD8+ T cells were found to play an important role in the observed protective antitumor effects generated by the vaccination. Similar results were observed using the HPV-16 E7 protein-based vaccination system. Thus, our data indicate that intramuscular administration of protein-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+ T cell immune responses. The clinical implications of the study are discussed.