Production of Human papillomavirus pseudovirions in plants and their use in pseudovirion-based neutralisation assays in mammalian cells

Abstract

Human papillomaviruses (HPV) cause cervical cancer and have recently also been implicated in mouth, laryngeal and anogenital cancers. There are three commercially available prophylactic vaccines that show good efficacy; however, efforts to develop second-generation vaccines that are more affordable, stable and elicit a wider spectrum of cross-neutralising immunity are still ongoing. Testing antisera elicited by current and candidate HPV vaccines for neutralizing antibodies is done using a HPV pseudovirion (PsV)-based neutralisation assay (PBNA). PsVs are produced by transfection of mammalian cell cultures with plasmids expressing L1 and L2 capsid proteins, and a reporter gene plasmid, a highly expensive process. We investigated making HPV-16 PsVs in plants, in order to develop a cheaper alternative. The secreted embryonic alkaline phosphatase (SEAP) reporter gene and promoter were cloned into a geminivirus-derived plant expression vector, in order to produce circular dsDNA replicons. This was co-introduced into Nicotiana benthamiana plants with vectors expressing L1 and L2 via agroinfiltration, and presumptive PsVs were purified. The PsVs contained DNA, and could be successfully used for PBNA with anti-HPV antibodies. This is the first demonstration of the production of mammalian pseudovirions in plants, and the first demonstration of the potential of plants to make DNA vaccines.

Figure 1. The pRIC3.0-mSEAP and pRIC3.0-iSEAP constructs and their replicons.

The top panel shows the pRIC3.0-mSEAP construct that recircularizes into the mSEAP replicon after the release of the T-DNA and the bottom panel shows the pRIC3.0-iSEAP construct. LIR, BeYDV long intergenic region; SIR, BeYDV short intergenic region, LB, left border; RB, right border, Rep (yellow), BeYDV rep gene; red vertical and semi vertical lines, the relative position where the T-DNA is transfected into the plant cells. The vector recircularizes at the duplicated LIR. The blue elements indicate the mammalian expression cassette and the green elements indicate the plant expression cassette.

Figure 2. Detection of PsVs in (a) dot blots of fractions 6–14, (b) L1 western blots of fractions 8–13, (c) L2 western blots of fractions 8–13 and (d) electron micrographs of HEK293TT cell produced PsVs and (e) electron micrographs of plant-produced PsVs.

The red boxes (in b,c) indicate the expected L1 and L2 sizes on the western blots, and the black arrows (in d,e) indicate the HPV particles. L, PageRuler Prestained Protein Ladder (Life Technologies) with sizes indicated; +, L1 or L2 positive controls, respectively, −, plant negative control.

Figure 3. Presence of replicon DNA in purified PsVs.

The diagram illustrates that a PCR product will only be present if recircularization of the replicon occurred. LIR, long intergenic region; arrows indicate primer positions. The bottom right part of the picture indicate the PCR products resulting from the inverse PCR; L, GeneRuler 1 kb DNA Ladder (Life Technologies); 1, mSEAP fraction 8 with proteinase K; 2 mSEAP fraction 8 without proteinase K; 3, mSEAP fraction 13 with proteinase K; 4, mSEAP fraction 13 without proteinase K.

Figure 4. The plant-produced PsVs were able to produce SEAP after pseudo-infection of mammalian cells.

SEAP expression was detected 3 days after HEK293TT cells were pseudo-infected with either SEAP plasmid DNA, plant produced mSEAP or iSEAP PsVs. Readings from untransfected/-infected cells were used as a baseline. Readings were done in triplicate. Error bars are indicated.

Figure 5. Pseudovirion-based neutralisation assay using the plant-produced PsVs.

Plant-made PsVs were pre-incubated with HPV-16 neutralising antibodies V5, E70, U4 and Gardasil antiserum and then used to infect HEK293TT cells for use of PBNA. Uninfected cells were used as a baseline and neutralizing activity was expressed as a percentage of neutralization compared to that of the average RLU readings of PsVs without antibody, which was set at 100%. The lighter shade of grey represents the SEAP signal that was reduced in the presence of the specific antibody, while the darker shade of grey represents the remaining SEAP signal post neutralisation. Error bars are indicated.