Date Published: June 18, 2018
Publisher: Public Library of Science
Author(s): Lea Bayer, Jessica Gümpel, Gerd Hause, Martin Müller, Thomas Grunwald, Robert D. Burk.
Papillomavirus capsids are known to have the ability to package DNA plasmids and deliver them both in vitro and in vivo. Of all known papillomavirus types, human papillomaviruses (HPVs) are by far the most intensely studied. Although HPVs work well as gene transfer vectors, their use is limited as most individuals are exposed to this virus either through a HPV vaccination or natural infection. To circumvent these constraints, we produced pseudovirions (PsVs) of ten non-human papillomavirus types and tested their transduction efficiencies in vitro. PsVs based on Macaca fascicularis papillomavirus-11 and Puma concolor papillomavirus-1 were further tested in vivo. Intramuscular transduction by PsVs led to months-long expression of a reporter plasmid, indicating that PsVs have potential as gene delivery vectors.
The transfer of nucleic acids for gene therapy or as genetic vaccines has several advantages, most importantly the rapid production, simple adaptation and high stability at ambient temperature of DNA. Additionally, the recipient’s cells themselves express the encoded antigens, allowing correct post-translational modifications and folding of the protein. Immunization with a DNA vaccine activates both the humoral and cellular immune response, making genetic vaccines a powerful platform . While intramuscular injection of naked DNA leads to a reasonable cellular uptake and subsequent expression in rodents , larger animals—especially non-human primates—require additional stimuli to enhance the uptake of the plasmid DNA . One of the most effective methods to enhance DNA-uptake is the use of electroporation [4,5]. However, electroporation is an invasive and painful procedure, requiring local anesthesia and the presence of special equipment . Other delivery methods include physical devices such as pressure injector, gene gun , and chemical formulations such as block copolymers , cationic liposome  and polyethyleneimine  and calcium nanoparticles . Furthermore, the application of a variety of bacteria and viruses as gene carriers has been explored, human papillomaviruses being one of them . Several characteristics of papillomaviruses make them promising candidates as DNA delivery vectors, such as their stability due to being non-enveloped, their ability to package foreign DNA up to 8kbp of without the need of a specific packaging sequence  and their capability to infect mucosal tissue . Gene delivery using papillomaviruses however does face the same issues as other more commonly used viruses: the problem of existing immunity against the vector.
Though not yet a competitive alternative to currently approved protein-based vaccines, gene-delivery through viral vectors holds great potential to increase the applicability of genetic vaccines. Due to the large amount of currently known NHPV types, these viruses provide the possibility to develop a large viral vector platform. Although cross-neutralization between different PV types is theoretically not expected, it remains to be elucidated in future studies to which degree cross-neutralization between antibodies developed by HPV- or NHPV-vaccinated individuals may restrict the gene transfer by NHPV PsVs. In this study we examined a range of NHPVs for gene delivery in cell culture and identified PcPV1 PsVs as effective for intramuscular delivery of a reporter gene. Gene sequences for the capsid proteins L1 and L2 of more than one hundred animal papillomaviruses are available in GenBank and can be used quite easily to produce pseudovirions. A main difficulty lies in the limited predictability of the suitability of the various types of PsVs to transduce a plasmid of interest: not only did we observe substantial differences in the transduction efficiency in vitro, but transduction in vivo must be tested for every individual papillomavirus type. Since transduction in vitro and in vivo has been shown for various PsVs based on genus α-HPVs , we speculated that this genus might provide especially suitable candidates for gene delivery. Interestingly, this did not prove to be the case. Of all tested PsVs of non-human α-papillomaviruses (CgPV1, PtPV1, MfPV6, MfPV11, MmPV1) only MfPV11 yielded good in vitro transduction rates. Upon intramuscular injection, however, MfPV11 PsVs showed only very week or no detectable transduction efficacy of the reporter plasmid and subsequent F.Luc-expression in vivo. A large number of HPV types belong to the genus of mucosa-infecting α-papillomaviruses, and α-HPV PsVs have indeed been successfully used for genital transmission in mice . Although this approach shows promise for mucosal application of papilloma PsVs as gene vectors, intense physical pretreatment of the mucosa was necessary, as the intact mouse genital epithelium was found to be quite resistant to infection with HPV16 PsVs [12,24]. The tropism of native papillomaviruses is thought to be primarily dictated by tissue specific enhancers, rather than by specific cellular entry receptors [25,26]. As many cell types have been shown to be susceptible to PV infection , it is therefore possible that the natural PV tropism has no bearing on in vivo transduction with PsVs. Intramuscular injection is a well-established and commonly accepted route for vaccine administration. We show here that this form of application could be a simple alternative for the administration of papilloma PsVs for gene transfer. As the PsVs were injected into the muscle, we assume that mainly muscle cells were transduced and expressed the reporter gene, although we did not check in detail, which cell type was transduced. Luciferase signals were observed only at the site of injection, but it would be of great interest to analyze whether an application to other organs would also be feasible.