One study compared the ability of a final protein boost with no final protein boost with respect to hybridoma generation and concluded that despite significant antibody responses in the immunized animals, the fusion of mouse spleen cells yielded a low number of and low-quality hybridomas unless the mice were given a boost 3C5 days before fusion.21 Other studies included an additional DNA plasmid immunization by intramuscular or intradermal injection 3C5 days before fusion as a final boost.42, 45, 51 Although the numbers of mAbs generated were small (that is, only a few mAbs from each fusion), mAbs with good binding affinity and diversity were reported.45, 51 The final DNA plasmid boost Propylparaben could also be delivered by hydrodynamic injection five days before fusion, and specific mAbs were successfully generated,25 including some against very difficult targets, such as multi-transmembrane proteins.27, 30 Proteins are commonly used as final boost reagents. delivery, DNA immunization, hybridoma, immunogenicity, monoclonal antibody INTRODUCTION The recent successful use of protective monoclonal antibodies as a life-saving treatment in Ebola virus-infected humans1 highlights the need for the development of new technologies that are fast and efficient in eliciting functional monoclonal antibodies (mAbs) to control emerging infectious diseases. DNA immunization as it exists today was pioneered in the early 1990s. Its initial use as a vaccination platform generated great enjoyment due to the overall simplicity of using DNA plasmids to deliver immunogens.2, 3, 4, 5 One particularly attractive feature of DNA vaccines is that immunogens are produced immunogen expression feature of DNA vaccines offers a number of benefits. Traditional protein-based immunization approaches have difficulty producing full-length protein immunogens by the recombinant protein method if the proteins are naturally expressed in a membrane-associated format, such as the multi-transmembrane G-protein coupled receptors (GPCRs) and ion channels. The DNA immunization approach can circumvent these problems because full-length proteins can be expressed when they are delivered in the form of DNA vaccines. Furthermore, it is well known that this structural integrity of proteins is critical for the induction of functional mAbs, yet these sensitive structures tend to be lost during the protein production process, regardless of whether they are produced as recombinant proteins or are extracted directly from cells or Propylparaben other sources in which the proteins are naturally expressed. Production of functionally active mAbs is usually highly dependent on the conformation of the proteins. Expressing intact immunogens by DNA immunization appears to have the very best chance of inducing mAbs with the desired biological activities. DNA vaccines possess the unique advantage of immunogen design flexibility. Immunogen inserts expressing the full-length sequences of target proteins Propylparaben are commonly used for all PSEN1 types of proteins, especially transmembrane proteins, with good success.19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 For intracellular proteins, one may assume that it is necessary to re-direct intracellular proteins into secretory pathways by adding a signal peptide to elicit a better antibody response. However, in a number of mAb production studies, native proteins have been used as DNA vaccine immunogen inserts without any sequence modifications.25, 28, 31, 32 On the other hand, the gene sequences of immunogen inserts for DNA vaccines can be easily edited to express designer proteins.’ For a single-transmembrane protein, the extracellular domain name of a secretory protein can be selectively cloned as the immunogen insert when the objective is to generate mAbs against epitopes around the extracellular domain name.33, 34, 35, 36, 37 Such an approach has also been applied to bacterial toxins. For example, a truncated fragment can be used for immunization in place of a full-length potentially lethal toxin protein, thereby avoiding the introduction of unwanted biological activity during the production of DNA vaccines, as well as during animal immunization.38, 39 Additional immunogen manipulations include the production of a mini-gene insert’ to express a short peptide sequence to cover a receptor-binding domain.40 In this case, antigenic determinants in the angiotensin-converting enzyme 2 binding domain of the severe acute respiratory syndrome spike protein, which does not closely match other coronaviruses, were predicted using software PROTEAN to induce anti-spike protein antibodies. Alternatively, a transmembrane anchor sequence can be added to non-membrane-associated antigens.41 As a simple and flexible immunogen design approach, DNA immunization offers a wide range of options to produce novel immunogen inserts for the induction of mAbs against even the most challenging targets (Table 1). Table 1 Types of DNA vaccine immunogens used for mAb induction has a major impact on the induction of high-quality mAbs. In one study of antibody generation via intravenous delivery of plasmid DNA,25 the relative efficacy of CMV promoter and the human ubiquitin C promoter was compared using luciferase as.
