Introduction

Antibodies remain a vital tool for the detection of proteins in basic research and diagnostics. The sequencing of ever more genomes continually increases the number of proteins that could be detected by specific antibodies. To generate such antibodies, conventional immunization methods require the isolation and purification of the protein of interest, an often difficult and time-consuming task that is distracting from the real research objective. Frequently, proteins cannot be isolated with sufficient purity or the antigenic determinant (epitope) is altered or lost during the isolation procedure. Alternatively, peptides can be synthesized to substitute for the protein, provided the immunogenic epitope is linear and not dependent on the tertiary structure of the intact protein. While useful in many instances, not all peptides can be synthesized cost-effectively and they lack posttranscriptional modifications, which are sometimes part of the epitope of interest. Genetic immunization strategies promise a general solution to these issues (1). Instead of delivering a protein or peptide to the host, an expression vector encoding the protein (or protein fragment) is introduced. The vector subsequently produces the protein in vivo. This avoids the requirement to isolate a protein, or synthesize a peptide, and produces a native protein with appropriate posttranscriptional modifications. This approach has been used extensively to invoke protective or therapeutic immune responses (“genetic vaccination”), using a variety of gene delivery methods, including direct injection of naked plasmid DNA into skeletal muscle, lymph nodes, or the dermis, electroporation, ballistic (gene gun) delivery, and viral vector delivery (2,3,4,5,6,7). An added advantage of plasmid DNA delivery is that bacte-rially methylated DNA can serve as its own adjuvant (8,9).

The successful use of genetic immunization (i.e., for the specific purpose of inducing an antibody response) has been previously described (10). These authors used a gene gun to deliver plasmid DNA expression vectors into mice and reported successful antibody production for 84% of tested antigens (detection of 50 ng of antigen using 1:5000 diluted immune serum by Western blot analysis). Of the existing gene delivery methods, direct injection of naked plasmid DNA is most attractive for genetic immunization purposes: excellent expression vectors are readily available, plasmid DNA production is easy and affordable (in contrast to viral vectors), and delivery is simple and does not require any equipment (in contrast to ballistic and electroporation methods). The disadvantage of direct intramuscular injection of naked plasmid DNA is the low transfection efficiency, especially in larger research animals (11). In recent years, two methods for the intravenous delivery of naked plasmid DNA have been described. Both rapidly deliver a large volume of plasmid DNA solution into the venous system, resulting in extravasation of the solution into the surrounding tissues and uptake and expression of the plasmid DNA. The first method delivers the plasmid DNA solution into the tail vein and transfects predominantly liver hepatocytes. This procedure is typically referred to as hydrodynamic tail vein (HTV) delivery (12,13). The second method delivers the plasmid DNA hydrodynamically into a limb vein (HLV delivery, trade-marked as Pathway IV™; Mirus Bio, Madison, WI, USA) and appears to transfect myofibers exclusively (14). In the present study, we evaluated the suitability of these intravenous plasmid DNA delivery methods for the induction of an antibody response by genetic immunization. Intravenous plasmid DNA delivery provides an advantage over alternative nonviral methods because it has a very high transfection efficiency and does not require specialized equipment.

The typical objective of genetic immunization is the isolation of polyclonal antibody containing sera or antibody-producing B cells, which can be used to generate monoclonal antibody (MAb)-producing cell lines (either by immortalization or by fusion with myeloma cells to form hybridomas). To evaluate whether an individual host has been effectively immunized, one typically uses the cognate immunization protein or peptide to screen sera for the presence of antigen-specific antibodies. The genetic immunization approach requires the generation of material for screening and confirmation purposes. Chambers and Johnston (10) introduced each gene of interest into two plasmid vectors: one placing the gene under transcriptional control of a mammalian promoter for genetic immunization in mice; another placing the gene under control of a bacterial promoter for the generation of protein in bacteria to be used for screening purposes. Not all proteins could be produced in bacteria (similar to the original problem of generating proteins for immunization purposes), and the benefit of mammalian posttranscriptional modification was lost. A more elegant solution to this problem is to use the same vector for both genetic immunization and antigen production. In these studies, we produced antigen in several mammalian cell lines transfected in vitro with the genetic immunization expression vector.