Immunisation with modified HPV16 E7 genes against mouse oncogenic TC-1 cell sublines with downregulated expression of MHC class I molecules
Introduction
Infection with human papillomavirus (HPV) can result in the development of a benign or a malignant tumour, the most serious HPV-associated disease being cervical carcinoma (CC) [1]. World-wide, CC is the second most common malignancy in women. However, most HPV infections are asymptomatic and transient and more than half of the untreated mild precancerous lesions regress spontaneously [2]. Immune surveillance seems to be responsible for HPV elimination, seeing that people with acquired or congenital immunodeficiency are at increased risk of CC. Accordingly, vaccination against the ‘high risk’ HPV types might suppress the development of HPV-induced diseases. Both therapeutic and prophylactic anti-HPV vaccines are being developed at the present time [3], [4], [5], [6]. While prophylactic vaccines should prevent HPV infection by inducing neutralising antibodies, therapeutic vaccines would be required to elicit cell-mediated immune responses capable of killing transformed cells. The only viral products consistently present in all transformed cells are oncoproteins E6 and E7 [7], which are necessary for both oncogenic transformation and maintenance of the transformed state. Therefore, they represent the targets of choice in the development of HPV-specific vaccines directed against CC.
Several different anti-HPV vaccines are being tested in animal models and in clinical trials [3], [6]. Because of many advantages, DNA immunisation represents one of the most promising approaches [8]. In order to increase the immunogenicity of the E7 oncogene, it has been modified in various ways and enhanced anti-tumour responses induced by E7-based DNA vaccines have been obtained. (i) The E7 gene has been fused with the sorting signals of lysosome-associated membrane protein 1 (LAMP-1; fusion gene Sig/E7/LAMP-1) [9], [10], [11], [12], the heat-shock protein 70 (Hsp70) of M. tuberculosis [13], the translocation domain of Pseudomonas aeruginosa exotoxin A [14], the extracellular domain of the Flt3-ligand [15], or the VP22 protein of herpes-simplex virus type 1 [16], [17]; (ii) E7 has been mutated in two zinc-binding motifs [18] or in the binding site for pRb tumour-suppressor protein (Rb-binding site) [19].
During tumour development, transformed cells are under the pressure of the host’s immune system. This leads to the selection of cells with adaptations that provide them with a survival advantage [20]. One of the mechanisms that enable cell escape from the immune system is reduction of the surface expression of the major histocompatibility complex (MHC) class I molecules [21]. This reduction has been recorded in up to 70–90% of CC patients [22], [23]. In patients at an early stage of tumour development, downregulated MHC class I production has been found associated with a worsened prognosis [24]. Moreover, metastatic cells have been shown to have lower MHC class I expression in comparison with cells from primary tumours [25].
A downregulation of MHC class I expression may be effected by several mechanisms, which give rise to distinct phenotypes [26], [27]. Defects in the antigen-processing machinery (APM) are often responsible for a low level of MHC class I surface expression [28]. Decreased levels of APM components have been demonstrated in many human [29], [30], [31], [32], [33], [34], [35] and mouse tumour cells [36]. Moreover, an association between the oncogenic transformation of cells and the downregulation of APM and MHC class I expression has been established [37], [38], [39].
At present, mouse oncogenic TC-1 cells are most often used in testing the efficacy of experimental therapeutic vaccines against the most common ‘high risk’ HPV type, i.e. HPV16, which is present in about 50% of CCs [40]. This cell line has been prepared by transformation of primary lung cells with HPV16 E6 and E7 oncogenes and activated H-ras [10]. The cells express a high level of MHC class I molecules. However, any population of cells forming a tumour is heterogeneous in MHC class I expression [41]. Therefore, an anti-tumour vaccine should induce immunity against tumour cells with a variable MHC class I production.
In this study, we derived clones of TC-1 cells with a decreased MHC class I expression. Some of the properties of these clones were tested and the mechanisms which might be involved in the MHC class I downregulation were partially analysed. The clones were used to verify the efficacy of DNA vaccination with the Sig/E7/LAMP-1 gene, which had previously been shown to induce a strong immune response against TC-1 cells [11], [12]. Mutations in the Rb-binding site that had been shown to improve the immunogenicity of E7 [19] were introduced into Sig/E7/LAMP-1 and the resulting gene, Sig/E7GGG/LAMP-1, was compared with Sig/E7/LAMP-1.
Section snippets
Plasmids
The construction of plasmids pBSC, pBSC/E7, pBSC/E7GGG, pBSC/L1E7, and pBSC/E7LAMP was described previously [19]. In brief, pBSC is an “empty” plasmid carrying the immediate early cytomegalovirus promoter. Downstream this promoter, the following genes: wild-type HPV16 E7 (pBSC/E7), mutated E7GGG [19] (pBSC/E7GGG), fusion L1ΔCE71-60 [42] (pBSC/L1E7), and fusion Sig/E7/LAMP-1 [9] (pBSC/E7LAMP) were cloned into the XhoI site. The immunogenicity of all these constructs had been tested in previous
TC-1 clones with downregulated MHC class I expression
To obtain TC-1 cells with downregulated expression of MHC class I molecules, we prepared cell lines from tumours formed in an immunisation/challenge experiment reported previously [19]. In that experiment, mice were immunised twice with plasmid pBSC/E7, pBSC/E7GGG, pBSC/L1E7, or pBSC/E7LAMP and challenged with 104 TC-1 cells. Tumours developed in all non-immunised control mice, but only in a portion of the immunised animals. Some of them were excised on reaching a diameter of 1.0–1.5 cm and 10
Discussion
From HPV16-transformed mouse TC-1 cells, which expresses a high level of MHC class I molecules, several cell clones with downregulated surface expression of these molecules were derived. The clones were obtained from a tumour that developed in a mouse immunised with an E7 gene-containing plasmid, i.e. under conditions favouring the selection of tumour cells with enhanced immunoresistance. The reduction of MHC class I expression was reversible, seeing that IFN-γ treatment led to its
Acknowledgements
We wish to thank Mrs. V. Navrátilová for technical assistance. This work was supported by Grant No. NC/5900-3 obtained from the Internal Grant Agency, Ministry of Health of the Czech Republic, by Research Project No. CEZ MZ 0023736001 of Institute of Hematology and Blood Transfusion, Prague, by Grant Nos. 312/99/0542 and 301/02/0852/A from the Grant Agency of the Czech Republic, and by the Czech Terry Fox Foundation.
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