Anti-cancer activity of plant-produced HPV16 E7 vaccine

Vaccine 25 (2007) 3018–3021

Anti-cancer activity of plant-produced HPV16 E7 vaccine Silvia Massa a,c , Rosella Franconi a , Rossella Brandi b , Antonio Muller b , Vadim Mett c , Vidadi Yusibov c,∗ , Aldo Venuti b a

c

ENEA, Italian National Agency for New Technologies, Energy and the Environment, BAS BIOTEC GEN, C.R. Casaccia, Via Anguillarese 301, 00123 Rome, Italy b Laboratory of Virology, Regina Elena Cancer Institute, Via delle Messi d’Oro 156, Rome, Italy Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Suite 200, Newark, DE 19711, USA Available online 19 January 2007

Abstract The E7 oncoprotein from Human Papilloma Virus (HPV) is an attractive candidate for anti-cancer vaccine development. In this study, we engineered HPV16 E7 coding sequence (wild type or mutagenized sequence, E7GGG) as fusions to ␤-1,3-1,4-glucanase (LicKM) of Clostridium thermocellum and produced in Nicotiana benthamiana plants using a transient expression system. Target antigens were purified and evaluated in mice for their potential as prophylactic and therapeutic vaccine candidates. Both fusion proteins induced E7-specific IgG and cytotoxic T-cell responses and protected mice against challenge with E7-expressing tumor cells. Furthermore, when administered after challenge, these plant-produced antigens prevented tumor development. © 2007 Elsevier Ltd. All rights reserved. Keywords: Human papilloma virus; Cancer vaccine; Plant-produced vaccine

1. Introduction The “high risk” human papilloma viruses (HPVs) are the primary etiologic agents of cervical cancer, which represents the main cause of cancer-related death in women in developing countries and the third leading cause of cancer-related death among women worldwide [1]. A prophylactic vaccine against HPV is now available (GARDASILTM , Merck) but, due to the long latency period between infection and onset of cancer, the benefits of prophylactic vaccination will not be visible for decades [2–4]. Thus, a therapeutic vaccine, targeting already infected individuals, is also required. Successful immunotherapy should induce specific cell-mediated immunity that would rapidly clear an established infection and provide protection against future exposure. Treatment of HPV-associated diseases will benefit from therapies that boost natural immune-mediated tumor defense mechanisms and that focus the immune response on the relevant tumor antigens. ∗

Corresponding author. Tel.: +1 302 369 3766; fax: +1 302 369 8955. E-mail address: [email protected] (V. Yusibov).

0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.01.018

HPV type 16 (HPV16) accounts for 50% of all HPVrelated cancer cases worldwide [2], therefore a vaccine specifically targeting this type of HPV is particularly desirable. The HPV16 oncoproteins (E5, E6 and E7) are responsible for the onset and maintenance of the transformed state and, therefore, represent appropriate targets for therapeutic vaccines [3]. Several therapeutic HPV-specific E7-based vaccine formulations have been tested in animal models and some have advanced into phase II and III clinical trials [4–6]. Many of these potential HPV therapeutic vaccines eliminate tumors in animal models and some evoke specific cell-mediated immune responses in early phase human trials. However, therapeutic vaccination has been limited by poor presentation of viral antigens that are expressed at low levels and by poor trafficking of effector T-cell populations to non-inflamed mucosal/skin sites. Therefore, the use of adjuvant has been crucial for therapeutic efficacy [7]. In this study plant-produced fusions of E7 and the E7 mutant E7GGG to Clostridium thermocellum ␤-1,3-1,4glucanase (LicKM) were assessed in mice. Due to its reduced transformation potential, E7GGG has been proposed as a safer candidate than E7 for vaccine development [8]. The

S. Massa et al. / Vaccine 25 (2007) 3018–3021

LicKM fusions induced E7-specific IgG and cytotoxic T-cell responses, protecting mice against E7-expressing tumors. In addition, these vaccine candidates prevented tumor growth in pre-challenged mice.

3019

days 0, 15, 30, 45 and 60. For both the prophylactic and therapeutic studies control animals were administered 10 ␮g of E. coli-produced E7 or E7GGG or plant-produced LicKM with or without Quil A. Tumor growth was monitored by palpation twice a week.

2. Materials and methods 3. Results and discussion 2.1. Production of E7 and E7GGG in plants The HPV16 E7 oncogene (GenBank accession number KO2718) was mutated using the Quikchange Site-Directed Mutagenesis Kit (Stratagene; La Jolla, CA) to give E7GGG with the following amino acid substitutions in the pRBbinding site of E7: D21G, C24G and E26G [8]. The LicKM carrier molecule for expressing target antigens in plants is described elsewhere [9]. Sequences encoding HPV16 E7 and E7GGG were cloned as in-frame internal fusions of LicKM to obtain LicKM-E7 and LicKM-E7GGG. These fusions included the signal sequence of Nicotiana tabacum PR1a protein at their N-terminus, and the 6xHis tag followed by the endoplasmic reticulum retention signal KDEL at their C-terminus. The fusions were cloned in the plant expression vector pBID4 [9] to give pBID4-LicKM-E7 and pBID4-LicKM-E7GGG. Each construct was introduced into Agrobacterium rhizogenes strain A4 and the resulting bacteria were inoculated into Nicotiana benthamiana. Five to seven days post infiltration, target antigens were purified by affinity chromatography and characterized by SDS-PAGE and immunoblot analysis. To provide control material, LicKM was similarly produced in N. benthamiana. The samples were quantified by densitometry using GeneTools software (Syngene; Cambridge, UK). 2.2. Evaluation of efficacy of plant-produced LicKM-E7 and LicKM-E7GGG Four to eight week old female C57BL/6 mice (Charles River; Como, Italy) were used in all experiments. For the prophylactic study 10 mice per group received 40 ␮g of LicKM-E7 or LicKM-E7GGG (equivalent to approximately 10 ␮g of E7 or E7GGG, respectively) subcutaneously (s.c.) with or without Quil A adjuvant (10 ␮g/mouse) on days 0, 14, 28, 42 and 76. Samples of sera from animals were collected on the day of each administration and assessed for the presence of E7-specific antibodies by ELISA. On day 49 two animals in each group were sacrificed to evaluate cell mediated immune responses. All remaining animals were then challenged by s.c. injection with 5 × 104 E7-expressing TC1 tumor cells [10]. For characterization of immune responses, ELISA, ELISPOT and spontaneous delayed-type hypersensitivity (DTH) assays were performed as previously described [11]. For the therapeutic study, 8–10 mice per group were inoculated s.c. with 5 × 104 E7-expressing TC-1 tumor cells 3 days prior to s.c. immunization with 40 ␮g LicKM-E7 or LicKM-E7GGG with or without Quil A (10 ␮g/mouse) on

3.1. Expression and purification of LicKM-E7 and LicKM-E7GGG produced in N. benthamiana LicKM, LicKM-E7, and LicKM-E7GGG were expressed in N. benthamiana leaves and the recombinant proteins were purified and subsequently analyzed by SDS-PAGE to reveal the predicted sized proteins of 28, 39 and 39 kD, respectively (Fig. 1). The estimated yields for LicKM-E7 and LicKME7GGG were approximately 400 mg/kg of fresh leaf tissue and the estimated yield for LicKM was approximately 1 g/kg. Antigenicity of the plant-expressed proteins was verified by immunobloting using antibodies specific to LicKM and E7 (data not shown). 3.2. Immunogenicity and prophylactic potential of plant-produced LicKM-E7 and LicKM-E7GGG In the presence of adjuvant all animals immunized with LicKM-E7 or LicKM-E7GGG or with E. coli-produced E7 or E7GGG mounted a specific IgG response (Fig. 1B), although in the absence of adjuvant LicKM fusions did not induce significant humoral responses (Fig. 1B). Since CD8+ cytotoxic T cells have a recognized role as effectors in anti-cancer responses, the induction of E7-specific CD8+ T cells was investigated by ELISPOT. In the presence of adjuvant, high numbers of IFN␥ secreting cells were detected in mice vaccinated with LicKM-E7 or LicKME7GGG, whereas significantly lower numbers of E7-specific CD8+ cells were found in mice vaccinated with E. coliproduced E7 or E7GGG and no IFN␥ secreting cells were detected in LicKM vaccinated mice (Fig. 1C). Interestingly, both LicKM fusions and E. coli-produced antigens induced low but significant numbers of spots in the absence of adjuvant, with the LicKM fusions giving the higher number of ELISPOT counts (Fig. 1C). It has been established that HPV-specific CD8+ T cells are protective against challenge with an E7-expressing tumor [6]. Therefore, the anti-tumor activity of the immune responses induced by plant-produced E7 candidate vaccines was evaluated by challenging vaccinated mice with TC-1 cells. In the presence of adjuvant, LicKM-E7 and LicKM-E7GGG each elicited tumor protection in 100% of animals, whilst E. coli-produced E7 or E7GGG only induced protection in 80 and 60% of mice, respectively (Fig. 1D). Surprisingly, 80% of animals immunized with LicKM-E7 in the absence of adjuvant were protected (Fig. 1D). By contrast, only 20% of animals immunized with LicKM-E7GGG or either

3020

S. Massa et al. / Vaccine 25 (2007) 3018–3021

Fig. 1. Characterization and efficacy of plant-produced vaccine candidates. (A) Coomassie stain of purified proteins separated by SDS-PAGE. Lane 1: molecular weight marker; lanes 2–6: BSA standards (0.25, 0.5, 1, 2.5 and 5 ␮g); lanes 8–10: 1, 2 and 3 ␮l of LicKM-E7; lanes 11–13: 1, 2 and 3 ␮l of LicKM-E7GGG. (B) E7-specific serum IgG responses. Data are presented as optical density values at 405 nm of 1:500 diluted sera. Data from individual animals are shown along with mean values. (C) ELISPOT analysis of splenocytes from vaccinated mice. Data are presented as mean number of spots ± S.D. per 2 × 105 splenocytes. Grey and black columns refer to cells stimulated with or without specific CTL E7 peptide, respectively. (D) Prophylactic vaccination against TC-1-induced tumors. Data are represented as percentage of tumor-free mice. (E) Therapeutic vaccination against TC-1-induced tumors. Data are represented as percentage of tumor-free mice.

E. coli-produced antigens in the absence of adjuvant were protected (Fig. 1D). No protection was observed in animals immunized with LicKM (Fig. 1D). 3.3. Therapeutic activity of LicKM-E7 and LicKM-E7GGG against HPV16 E7-expressing tumors Animals that had been inoculated with E7-expressing TC-1 cells were subsequently immunized with LicKM,

LicKM-E7 or LicKM-E7GGG or with E. coli-produced E7 or E7GGG. All animals immunized with LicKM developed tumors within 4 weeks, whilst those treated with LicKM-E7 or LicKM-E7GGG plus adjuvant remained tumor-free for the duration of the study (10 weeks). In contrast, immunization with E. coli-produced E7 or E7GGG plus adjuvant inhibited tumor growth in only 40 and 60% of animals, respectively (Fig. 1E). In the absence of the adjuvant only LicKM fusions inhibited tumor growth, with greater protection observed

S. Massa et al. / Vaccine 25 (2007) 3018–3021 Table 1 Delayed type hypersensitivity ear thicknessa ± S.D. 48 h LicKM E. coli E7 LicKM-E7 LicKM-E7 + Quil A

2.5 2.3 5.0 6.5

72 h ± ± ± ±

0.7 1.5 2.7 0.7

1.0 4.6 10 7.0

± ± ± ±

0.7 2.5 2.3 0.1

S.D., standard deviation. a Ear swelling was reported as the mean of the differences () in thickness between challenged and unchallenged control ears from five mice per group (mm ear thickening × 10−2 ).

in animals that received LicKM-E7 (80%) versus LicKME7GGG (60%), whereas only 20% of animals treated with E. coli-produced E7 or E7GGG remained tumor-free (Fig. 1E). 3.4. Induction of a delayed-type hypersensitivity response by LicKM-E7 Protection against the development of HPV-associated disease is in large attributed to the cell-mediated immune responses. The DTH response is thought to represent antigen-specific cytokine mediated inflammation, particularly involving Th1 type cytokines. Since LicKM-E7 showed greater therapeutic activity than LicKM-E7GGG against E7expressing tumors, we evaluated the DTH response to E7 in mice vaccinated with LicKM-E7 or E. coli-produced E7. An antigen-specific DTH response was observed in mice vaccinated with the LicKM-E7 protein, even in the absence of adjuvant (Table 1). This response exceeded that observed in mice vaccinated with E. coli-produced E7. Mice immunized with LicKM showed no significant ear swelling, demonstrating that the LicKM carrier molecule does not induce an inflammatory response. In a previous study, crude plant extracts containing HPV16 E7 expressed using a potato virus X-derived vector were shown to provide 40% protection against challenge with an E7-expressing TC-1 cell line [11]. These experiments were extended in the present study where E7 and E7GGG were produced in plants as fusions to LicKM and the purified proteins were evaluated as prophylactic and therapeutic vaccine candidates against HPV-induced tumors. Taken together, the induction of E7-specific IgG along with DTH and ELISPOT data indicate that the plant-produced LicKM-E7 and LicKM-E7GGG fusions are highly immunogenic, inducing both humoral and cell-mediated immune responses, superior to those generated by immunization with E. coli-produced E7 and E7GGG. These data were reinforced by results demonstrating that both LicKM-E7 and LicKM-E7GGG were more active than E. coli-produced E7 in inhibiting tumor growth in the therapeutic study. Addi-

3021

tionally, when the vaccine preparations were administered without adjuvant, only the LicKM fusion proteins were able to prevent tumor growth suggesting that these LicKM fusion proteins alone are able to activate both innate and adaptive antigen-specific immune responses. Further studies are being conducted to characterize the potential role of LicKM in the enhanced immunogenicity and protective efficacy of fusion vaccine candidates. In conclusion, the reported results support the concept of producing an anti-tumor vaccine with both therapeutic and prophylactic potential in plants.

Acknowledgements The authors thank Dr. G. Fernando for kindly providing Quil A. The authors also thank Margaret Shillingford for care and maintenance of plants, and Dr. Stephen Streatfield and Dr. Jessica Chichester for critical reading and editing of the manuscript. Work partially supported by the Italian Ministry of Health.

References [1] Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999;80(6):827–41. [2] Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995;87(11):796–802. [3] Fehrmann F, Laimins LA. Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation. Oncogene 2003;22(33):5201–7. [4] Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB, et al. A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 2002;347(21):1645–51. [5] Billich A. HPV vaccine MedImmune/GlaxoSmithKline. Curr Opin Investig Drugs 2003;4(2):210–3. [6] Frazer IH. Prevention of cervical cancer through papillomavirus vaccination. Nat Rev 2004;4(1):46–54. [7] Gerard CM, Baudson N, Kraemer K, Bruck C, Garcon N, Paterson Y, et al. Therapeutic potential of protein and adjuvant vaccinations on tumour growth. Vaccine 2001;19(17–19):2583–9. [8] Smahel M, Sima P, Ludvikova V, Vonka V. Modified HPV16 E7 genes as DNA vaccine against E7-containing oncogenic cells. Virology 2001;281(2):231–8. [9] Musiychuk K, Stephenson N, Bi H, Orozovic G, Brodelius M, Brodelius P, et al. A launch vector for the production of vaccine antigens in plants. Influenza Other Respir Viruses, in press. [10] Lin KY, Guarnieri FG, Staveley-O’Carroll KF, Levitsky HI, August JT, Pardoll DM, et al. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 1996;56(1):21–6. [11] Franconi R, Di Bonito P, Dibello F, Accardi L, Muller A, Cirilli A, et al. Plant-derived human papillomavirus 16 E7 oncoprotein induces immune response and specific tumor protection. Cancer Res 2002;62(13):3654–8.