Elsevier

Vaccine

Volume 21, Issues 7–8, 30 January 2003, Pages 649-658
Vaccine

Induction of immune responses by DNA vaccines in large animals

https://doi.org/10.1016/S0264-410X(02)00574-1Get rights and content

Abstract

It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection deficiency and the low level of expression elicited by plasmid vectors in large animals. In our attempt to enhance transfection efficiency and, thereby, enhance immune responses, we employed a variety of methods inducing gene gun delivery or suppositories as delivery vehicles to mucosal surfaces, as well as electroporation for systemic immunization. To test these different systems, we used two different antigens—a membrane antigen from bovine herpesvirus glycoprotein (BHV-1) gD and a particulate antigen from hepatitis virus B. Gene gun and suppository delivery of BHV-1 gD to the vagina resulted in the induction of mucosal immunity not only in the vagina, but also at other mucosal surfaces. These data support the contention of a common mucosal immune system. In the case of electroporation, we were able to develop significant enhancement of gene expression following electroporation with surface electrodes (non-invasive electroporation) as well as invasive electroporation using single or six-needle electrodes. Various delivery systems such as bioject or needle delivery also influenced the immune response in both the presence and absence of electroporation. These studies also demonstrated that co-administration of plasmids coding for two different antigens (BHV-1 gD and hepatitis B surface antigen (HbsAg)) did not result in significant interference between the plasmids. These studies suggest that various combinations of delivery systems can enhance immunity to DNA-based vaccines and make them practical for administration of these vaccines in large animals.

Introduction

It has been just over 10 years since the first reports regarding genetic (polynucleotide) immunization were published [1], [2], [3]. The original excitement of developing vaccines using this technology even heralded the technology as the “third generation of vaccinology” [4]. As a result, over 1000 different reports of this technique have been published with antigens from different bacteria, viruses, and parasites (reviewed in [5], [6]). Unfortunately, the majority of the most successful demonstrations of the efficacy of DNA immunization have been performed in the mouse. When similar approaches were used in humans or large animals the results were not as encouraging. Although polynucleotide vaccines have not been very efficient in large animals or in humans there continues to be an interest in developing this technology for these species since DNA vaccination has many advantages over conventional vaccines. These advantages include the endogenous production of antigen. This is a tremendous advantage, especially for viral antigens where all the post translation modifications are similar following DNA immunization as those present during infection. As a result, the antigens are authentic with all the conformational epitopes required for protection being expressed. A second advantage is that since the animal acts as a bioreactor, there is no need for downstream processing of the vaccine after the plasmid is purified. Antigen purification is often a laborious and expensive process. Thus, DNA-based vaccination should be economical to produce. Since the antigen is produced endogenously there is also no need for adjuvants or problems associated with injection site reactions produced by adjuvants [7]. Because of the endogenous expression of antigens, plasmid-based vaccines induce a more balanced Th1/Th2 like immune response which is important for clearance of many viral infections [8]. Possibly one of the major advantages of DNA vaccines is that they can induce immune responses in neonates even in the presence of passive antibody [9], [10]. Recently, we have demonstrated that it is even possible to induce immunity in utero [11]. This has the real advantage of insuring that individuals are born immune to pathogens which they may experience early in their life. For example, transmission of herpes simplex, hepatitis B, human immunodeficiency virus, Group B Streptococcus, Haemophilus influenzae type B, and Chlamydia sp. occurs shortly before, during, or after birth. To reduce the risk of vertical disease transmission of these diseases, Cesarean sections, antibiotic treatments, or maternal antiviral therapy during the last trimester are used where available, together with improved neonatal care. Unfortunately, none of these approaches completely eliminates the risk of neonatal infection. Therefore, there is a need for developing vaccines that could be employed in these instances. Finally, since DNA vaccines are simple to purify, and technologies are available for purification the risk of extraneous contaminating agents, which are a major problem in conventional live vaccines is eliminated [12], [13].

The reasons for the lower efficacy of DNA vaccines in humans or large animals are not currently known, but it could be related to the transfection efficiency. In mouse cells, transfection appears to occur with much more efficiency than in larger animals species. Thus, improving delivery of the plasmid to enhance cellular transfection and subsequent expression of proteins will be a critical factor in developing vaccines for large animals and humans. The present report describes the progress made in the last few years in addressing some of the factors influencing the efficacy of DNA vaccines, specifically focusing on improving transfection efficiency and gene expression by improved vectors and delivery systems.

Section snippets

Enhancing antigen expression

The simplest concept of a DNA based vaccine incorporates a promoter, the gene of interest for use in the vaccine, and a backbone for delivery of the cassette into cells. Manipulation of each of these different components of the plasmid has been shown to alter the efficacy of DNA vaccination. Since it is generally believed that the level of gene expression should correlate with the level of immunity, the plasmid construct should be one that maximizes gene expression in vitro. If the transfection

Delivery

Although it is difficult to quantitate the number of plasmids that enter cells or are degraded before they enter the nucleus and initiate gene expression it is believed that in excess of 90% of the DNA never gets into the cytoplasm and of this 10% less than 1% enter the nucleus where gene expression occurs [39], [69]. Thus, there have been numerous approaches used to enhance plasmid uptake not only into the cell but into the nucleus. The earlier approaches focused on lipid based delivery

Mucosal delivery

Since the majority of pathogens enter through mucosal surfaces of the gastrointestinal tract, respiratory and reproductive tracts, delivery of vaccines to induce mucosal immunity would be a major achievement [89], [90]. Thus, if it was possible to prevent infection, by neutralizing the pathogen at the site of entry, this would be more effective than preventing disease after infection, as is the case with many vaccines given parenterally. Secondly, by inducing mucosal immunity, the amount of

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