There is a need for therapeutic HPV vaccines that eliminate existing lesions and malignant tumours by inducing cell-mediated immune responses against HPV-infected cells. Presently, various strategies such as DNA based, peptide- and protein based and live-vector based vaccines are utilized . DNA vaccines have emerged as potentially useful HPV therapeutic vaccines, but there is limited success after clinical trials of DNA vaccines so far and in the actual commercialization of the product. Moreover, the fear of DNA integration into the host genome and subsequent genomic instability remains. Therapeutic protein vaccines, on the other hand, are generally considered safe. One drawback of protein vaccines is that they are often not very immunogenic, and often need either to be fused to other immunogenic proteins, or have adjuvant added, in order to increase their immunogenicity. Production of protein vaccines can also be very costly when it is done in mammalian cells and the concern of contamination by other human or human-infecting viruses remains. Plant-produced proteins could provide an alternative as they can be produced economically and have been shown to be safe for use in humans .
In this study we investigated if the plant-produced shuffled HPV16 E7 protein fused to Zera® is a suitable candidate as a therapeutic vaccine for HPV-16 infections and HPV-related tumours. An artificial shuffled HPV-16 E7 gene (16E7SH) was selected as it has previously been shown to cause regression of tumours in mice when tested as a DNA vaccine. 16E7SH was fused to Zera®, a novel signal sequence which promotes the formation of protein bodies during expression of fusion proteins. We wanted to investigate the role of Zera® in increasing 16E7SH production in plants, and also its role in enhancing the immunogenicity of the plant-produced protein. Lastly, the possibility that free Zera® could act as an adjuvant was explored by mixing Zera® PBs with the purified 16E7SH protein.
For safety reasons the use of wildtype HPV-16 E7 for vaccination is not feasible in humans. Approaches like the introduction of point mutations into the E7WT gene, however, lead to an unwanted loss of naturally occurring epitopes that is potentially associated with a decrease in vaccine efficacy. We used a rearranged (“shuffled”) E7 sequence which lacks transforming properties . Ultimately this non-transforming HPV-16E7SH supplies all potential naturally-occurring T cell epitopes, covering the broad range of MHC restriction. Consequently, prior knowledge of the patient’s HLA-haplotype is not required which is especially important in the outbred human population. In addition, a more potent immune response may be induced, involving all occurring HLA-restriction elements in the vaccine.
We tested whether 3 different recombinant HPV16 E7-derived proteins could be produced in plants. The expression in plants of recombinant protein from constructs encoding 16E7SH alone, 16E7SH fused to Zera®, and Zera® fused to wildtype HPV-16E7, was assessed by comparing expression at 3, 5, 7 and 10 dpi. ZERA-16E7SH and ZERA-16E7 proteins were successfully expressed in plants as 29 and 24 kDa fusion proteins, respectively. However, the expression of 16E7SH alone was not detected, indicating that fusion with Zera® enhanced accumulation levels of the protein in plants. The incorporation of a silencing suppressor increased ZERA-16E7SH accumulation by 6-fold, indicating that post-transcriptional gene silencing (PTGS) plays a role in the expression of the proteins.
This increase with addition of a silencing suppressor is lower than other reported increases, such as the 30-fold increase measured using with GFP expression . However, the accumulation of ZERA-eGFP reached ±25 g/kg in our experiments (data not shown), which was more than 70-fold higher than the GFP expression reported by Voinnet et al. .
The Zera®-HPV proteins were expressed at levels ranging from 0.1 - 6 g/kg. ZERA-16E7SH levels were the highest, varying from 1 - 6 g/kg. This was 2-fold higher than that obtained by Massa et al. , who attained levels of 0.4 g/kg for E7 fusion protein production in N. benthamiana. In contrast, expression of the 16E7SH protein alone was too low to quantitate, due either to a very poor level of expression, and/or to degradation during extraction.
The time trial expression profiles showed stable protein accumulation up to 12 dpi in samples infiltrated with ZERA-eGFP and for the ZERA-16E7 constructs. This is much longer than seen in previous studies where the expression of the proteins peaked at 60-70 h, even with the addition of a silencing suppressor . Protein degradation in the cytoplasm is one of the reasons behind this decreasing protein concentration, which is alleviated by the sequestration of the proteins into PBs. As the Zera® fusion protein is translated, it is directly sequestered into ER-derived and membrane-delimited protein bodies. It is thought that this encapsulation protects the fusion protein from proteolytic degradation. At the same time, the PBs serve as a very useful means for purification of the proteins, as they are dense organelles which can be easily purified on density gradients .
The ability of the plant-produced ZERA-16E7SH PBs to cause tumour regression was shown to be significant and similar to that of the DNA vaccine equivalent. Further co-inoculation of ZERA-16E7SH PBs with adjuvant, however, did not significantly enhance tumour regression suggesting that Zera® has an adjuvanting effect by itself. Inoculation of tumourigenic mice with plant-produced ZERA-eGFP lacking 16E7SH also did not result in tumour regression suggesting that Zera is not immunogenic. Despite the fact that it has been shown that eGFP is minimally immunogenic in C57/BL6 mice , the lack of tumour regression observed when mice were inoculated with ZERA-eGFP further supports the evidence that the 16E7SH is the immunogen causing tumour regression and that Zera® does not contribute to this.
The pTH-ZERA-16E7SH construct appeared able to induce higher immune responses than the Zera®-free counterpart (pTH-16E7SH); however, the data were not in all cases statistically significant. In general, the DNA-based vaccines were more efficacious than the protein-based vaccines in the context of control of tumour growth (Figure 5), which probably reflects in part the fact that DNA vaccines induce more Th1 than Th2 responses after intramuscular injections. The pTH-16E7SH construct did, however, also induce a moderate IgG response, as measured by ELISA (Figure 7). Additionally, IFN-γ levels of splenocytes isolated from mice inoculated with pTH-ZERA-E7SH were elevated compared to mice inoculated with pTH-16E7SH (Figure 6). This trend was also observed in Granzyme B ELISPOT assays as well as in chromium release assays.
One way to enhance potencies of DNA vaccines is to increase antigen expression in professional antigen presenting cells, such as dendritic cells. As ZERA-16E7SH is expressed well in plants, and 16E7SH is not, it can be speculated that ZERA-16E7SH is also expressed to much higher levels from a DNA vaccine in mammalian cells than 16E7SH alone, and that this would in turn enhance CTL response induced by this DNA vaccine . In fact, the increase in protein accumulation due to fusion with Zera® and production of PBs has been observed in mammalian cells as well as other organisms . It can be speculated that PBs are also formed in the mammalian cells that are inoculated with the DNA vaccine. It remains unclear how such heterologous organelles, which typically are retained in the ER, could in turn enhance the immune response as we observed in this study.
In our study we showed that Zera®-PBs were clearly able to stimulate humoral and cellular immune responses either as ZERA-16E7SH fusion protein or as Zera® PBs co-inoculated with 16E7SH proteins. Normally, subunit protein vaccines are not very efficient in the induction of the cellular immune responses; thus, this finding could be of great interest in the context of protein-based therapeutic vaccines. It is well known that professional antigen presenting cells like DCs and macrophages favour the uptake of particles with repeating sequence motifs. The adjuvanting effect of Zera® could also be due to its particulate nature. Interestingly, in our hands IFA was in no case able to enhance immune responses significantly in the presence of Zera®, which was wholly unexpected. The same IFA lot used in the present study induced an enhancement of the antibody response after immunization with other antigens (highly purified recombinant OVA (data not shown)). In addition, despite the fact that IFA is commonly used to enhance Th2-directed responses, we observed in the OVA immunizations an effect on the Th1 response.