A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses

Objectives Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. Results In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E.coli (BL21). The puri�ed protein was con�rmed by SDS page and western blotting and then injected into the C57BL/6 mice. The e�ciency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD+8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specic CD8+ T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. Conclusion Our �nding suggested that this novel fusion protein vaccine was able to induce therapeutic e�cacy and immunogenicity by improving CD8+ T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.


Introduction
Human papillomavirus infection (HPV) is the most common viral infection which is causes cancer or genital warts (Haghshenas, Mousavi et al. 2016).Based on the location of HPV lesions, various types of cancer such as cervix, anal, vagina, throat, vulva, penis, and breast could be developed.More than 200 types of HPV have been identi ed, of which 40 types are belong to genital areas (Haghshenas, Mousavi et al. 2017) (Mousavi, Ra ei et al. 2020).HPV types are divided into high and low risk groups.Low-risk types such as 6 and 11 lead to 99% of genital warts and laryngeal papillomatosis; while 70% of cancers are related to high-risk types, such as 16, 18 (Akhondnezhad, Haghshenas et al. 2018).The HPV virus belongs to papillomaviridae which is encoding two structural (L1, L2) and nonstructural protein (E1, E2, E4, E5, E6, and E7).E6 oncoprotein, is a key factor in tumor progression, act as P53 suppressor through binding to its C-terminal region.This protein is a transcription factor that controls cell proliferation by preventing the G 1 to S phase (Gulati, Huang et al. 2018).E7 oncoprotein is a small nuclear protein with no enzymatic activity.This protein is bound to the hypophosphorylated retinoblastoma protein (PRb); in this situation, the cell cycle moves to the S phase.Then, DNA synthesis and cell proliferation takes place (Münger, Androphy et al. 2018).Regarding the importance of E6 and E7 in induction and maintenance of cellular transformation, these early genes have been reported as suitable targets for designing anti-tumor vaccines and controlling HPV infection (Li, Chen et al. 2017) (Tang, Yin et al. 2012).
In particular, dendritic cells (DCs) are one of the most important APCs and play an important role in immune response against viral infections (Eagar and Miller 2019).DCs function, macrophagein ammatory protein-1α (MIP)-1α, MIP-1β and RANTES genes activation is associated with the high expression of MHC-I, CD40, CD80, CD86 and production of IL-1β, TNF-α, IL-12, IL-15 cytokines (Fanales-Belasio, Moretti et al. 2002).The serious problem associated with protein and peptide vaccines is that the structure of peptide antigen could be altered and do not elicit strong immune response (Du, Liang et al. 2020).On the other hand, these proteins enter the cells via an endocytic and activate the MHC-II presentation pathway; so they are unable to induce strong cytotoxic CD8+ T cell response (Blum, Wearsch et al. 2013).Today, there are several ways are used to transfer protein in to the cell.Cell penetrate peptides (CPP) such as Tat, Pep-1, LAL, Cady-2, P28, hpp10 peptide with potential interact to proteins and peptides are able to transfer different types of cargo (protein and peptides) into the cell (Shahbazi and Bolhassani 2018).It is suggested that a novel delivery system, Tat protein (small and soluble protein with 86-101 amino acids), with high e ciency and low cytotoxicity is able to transport peptides and proteins into cytoplasm via the plasma membrane and it is well-suited for the delivery of antigenic peptide to MHC-1 presentation pathway (Zou, Peng et al. 2017).Study shows that vaccination with Tat protein provides protection against viral replication, which can induce Th1 immune response as well as the Cytotoxic T lymphocytes (CTLs) response (Alipour, Mahdavi et al. 2017).
Prophylactic and therapeutic vaccination is highly effective in control and prevention of HPV infection.
Two prophylactic HPV vaccines (Gardasil, Cervarix) are composed of L1 virus-like particles (VLPs) and they are used for the prevention of HPV infection and cervical cancer (Barra, Leone Roberti Maggiore et al. 2019).In the current study, we designed a novel therapeutic vaccine based on E7 protein of HPV16,18,6,11 and Tat peptide against cervical cancer. HPV16,18,6, protein was assessed in vitro and examined for induction of E7-speci c humoral and cell-mediated immune responses in C57BL/6 mice model.This study shows how a novel fusion protein induce therapeutic e cacy against HPV16-positive tumor cells in mice.
Whole sequence was synthesized by Biomatik, (Cambridge, Canada) and cloned into His-tag harboring pET-22b vector.In order to establish an additional fusion protein by the lack of Tat sequence, Nco1 and Xho1 restriction sites were placed between the chief protein (E7-TAT) and then cloned to pET-28a vector.The plasmids pET22b-E7-TAT and pET28a-E7 were puri ed.

Production of fusion proteins (E7-TAT, E7)
Expression plasmids (pET-22b , pET-28a) were transformed to the E.coli (BL21) and cultured on LB broth containing speci c antibiotics, and then incubated at 37°C by shaking 170 rpm.The OD was checked until the optimal density reached to 0.6-0.8 at 600nm.One milliliter of the culture without inducer was used as a negative control.The culture was induced by adding 0.1-0.5 mM of Isopropyl-D-thiogalactoside (IPTG) and incubated at 37°C for 16 h.Harvest bacterial cells were centrifuged at 6000g at 4°C for 15 min; then the supernatant discarded and cell pellet frozen at -20°C.After re-suspending pellet in the buffer solution, it is found that the expressed proteins aggregated in inclusion bodies.The next day bacteria were re-suspended in buffer [50mM Tris-HCL PH=7.5, 1.5M NaCl, 20mM Imidazol, Urea 8M, Guanidine 6M, 5mM 2ME and 2% Triton100x ].Then cells were sonicated on ice for 3 min with 30 sec interval.The mixture then centrifuged at 6000g at 4°C for 15 min and the soluble fraction was transferred to a new 15ml tube.At the nal, 500mM Imidazol and a nity chromatography based on Ni-NTA (Qiagen) column were used for puri cation of the recombinant fusion protein using 6xHis-tag.

SDS page and western blotting analysis
After puri cation of recombinant fusion protein, dialysis was performed for the removal of residual salts, then concentration of protein was assessed by BCA kit (DNA Biotech, Iran) according to the manufacturer's instructions.After that, to con rm of the presence of puri ed protein, the fraction was analyzed by the 12% SDS-PAGE gels (w/v) and then transferred to a PVDF membrane for 2h by Bio-Rad transfer system.PVDF membrane was shacked by TTBS (Tween 20 0. 1%, Tris base 1M, NaCl 0.9%) and 5% skimmed milk overnight.After washing with TTBS three times, the membrane treated with mouse anti-His-tag antibody (Thermo Fisher Scienti c,US) at 1:1000 dilutions at room temperature (RT) by shacking 2h, and then washed with TTBS three times.The membrane was treated in the secondary HRPconjugated antibody (Thermo Fisher Scienti c,US) at 1:5000 dilutions by shacking 2h at RT; then it was washed with TTBS three times.The result was analyzed by staining with ECL and Luminescence Image System (Hansor, Taiwan).

Cell culture
TC-1 cells (expressing HPV16 E6 and E7 and generated by co-transformation of C57BL/6 mouse lung epithelial cells), KB cell (contain human papillomavirus18 (HPV18) and Caski cell (contain human papillomavirus16 (HPV16) sequences) were purchased from the Pasteur Institute of Iran.TC-1 cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotic in a humidi ed atmosphere of 5% C02.Caski and KB were cultured in Roswell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (FBS) and antibiotic in a humidi ed atmosphere of 5% C02.

Preparation of cell lysate
KB and Caski cells were De-attached and washed with PBS buffer for three times.The cells were centrifuged at 10000g for 10 min.After that the pellet were subjected to freeze-thaw between liquid nitrogen and 37˚C water bath for 3-4 times.The amount of total protein was measured by BCA kit according to the manufacturer's instructions.In present study, HPV6 and HPV11 cell lysate were obtained from clinical sample.

Mice immunization
Inbred female C57BL/6 mice (8-12 weeks old) were purchased from the Pasteur Institute of Iran and maintained under speci c pathogen free conditions.Six groups of ve mice (n=5) were subcutaneously injected with 20 µg/ml E7-Tat, 20 µg/ml E7-Tat plus Freund's adjuvant (50: 50 v/v), 20 µg/ml E7, 20 µg/ml E7 plus Freund's adjuvant (50: 50 v/v), PBS and Freund's adjuvant at the right ank three times on days 0, 14, and 28.Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were emulsi ed with protein for the 1st and 2nd respectively.For analysis of speci c IgG antibody assay, the blood samples were obtained before vaccination, two weeks after the second vaccination and one week after the last vaccination.(Table 1) (Fig 1).

Antibody response assay
Mice were sacri ced 10 days after the third immunization and the level of speci c IgG antibody was measured by ELISA for detection of antibodies to E7 and E7-Tat.Brie y, 10 µg of E7 and E7-Tat protein were coated with PBS buffer in 96 well at-bottom ELISA plate (Nunc), overnight at 4°C.After washing with PBS buffer and Tween 20 (5%) for 3 times, wells were blocked with % BSA (bovine serum albumin) for 2 h at 37°C.Then, after washing, serum samples were diluted 1:100 to each well in triplicate and incubated for 1.5 h at RT.After washing, a goat anti-mouse IgG HRP antibody (diluted 1:10,000 in 1% BSA/PBS-Tween) was added to each cell, and the plate was incubated for 1.5 h at RT.The plate was washed and TMB solution was added to each well.After 10 min, the stop solution (H2SO4 (2N)) was added and the absorbance was measured at 450 nm.

MTT assay for toxicity of HPV6 and HPV16 cell lysate
The MTT assay was used for evaluation of the toxicity of the clinical and cell lysate samples (HPV6 E7 and HPV16).Brie y, normal C57 BL/6 splenocytes (500,000/well in RPMI 1640 containing 10% FBS) were seeded onto 96-well culture plates in triplicate.After 24 h of culture, cells were stimulated with different doses of protein (100 µg/ml, 50 µg/ml, and 20µg/ml) and incubated for 48 h.After incubation the supernatants were removed and the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide], Sigma, 5 mg/mL in PBS) was added and then cells were incubated at 37°C in 5% CO2 for 4-6 h.The absorbance of purple formazan crystal was measured at 590 nm by an ELISA reader after dissolution in dimethyl sulfoxide (DMSO), Sigma.

cytokine assay (IL-4 and IFN-γ)
After sacri cing the mice, the spleen of each mice was isolated.A total of (5 × 10 5 /ml) splenocytes of mice in each treatment group were stimulated with 100µg /ml concentrations (Kim, Hong et al. 2006) of HPV6, HPV11, KB (HPV18), Caski (HPV16) cell lysate and 10µ/ml of each puri ed protein (HPV E7 and HPV E7-Tat) in RPMI 1640 medium supplemented with 10% FBS and 100 µg of streptomycin per ml for 48 h in a 37 C • , 5% CO2 incubator.After that supernatant was collected for the presence of cytokines IL-4 and IFN-γ.Concentration of cytokines were determined by speci c sandwich ELISA-Reader according to the manufacturer's instruction.(Invitrogen, United States) with C.N 88-7044 (Mouse IL-4 Uncoated ELISA) and 88-7314 (Mouse IFN gamma Uncoated ELISA).

CD+8 cytotoxicity
Splenocytes from each mouse harvested in cell medium at 5 × 10 5 /ml and stimulated with 100µg /ml concentrations of HPV6, HPV11, KB, Caski cell lysate and 10µ/ml of each puri ed protein (E7 and E7-Tat) for 48 h in a 37 C • , 5% CO2 incubator.Then the pellet were collected and labeled with FITC-conjugated anti-CD8a and PE-conjugated anti-CD107a antibody (invitrogen, USA) for cytotoxicity activity.We used isotype matched antibodies as control (Invitrogen, USA).Data were analyzed using Flowmax software (TreeStar, San Carlos, CA).

Statistical Analysis.
Statistical analysis was assessed using ANOVA (one-way and two-way) followed by Tukey's test (Graph Prism Pad version 8.0.1 soft-ware).Survival was analyzed using the log-rank (Mantel-Cox) test.This result considered statistically signi cant as a P value of <0.05.

Expression and puri cation of the recombinant fusion
The plasmid pET22b E7-Tat vector was ed transformed into BL21.Fusion protein expression was induced by adding IPTG to LB broth media.For optimization of the protein expression, some various conditions were tested, such as temperature, concentration of IPTG, induction time of expression and host.This study shows that induction with 0.1-0.5 mM IPTG at 37°C for 16 h is suitable condition for expression of the fusion protein (Fig. 4.A,B).The sonicated cell supernatants were puri ed using Ni2+ (Ni-NTA super ow) a nity chromatography.Puri cation of HPVE7-Tat and HPVE7 protein with the molecular weight of 48.57and 46.68 KDa is shown in Fig. 5 (A,B).The puri ed recombinant protein transferred to PVDF membrane for western blotting analysis then, they were detected by anti-His antibody (Fig. 6. A,B).According to the BCA protein assay kit, the total concentration of protein was calculated 600 /ml.

Protective response elicited in E7-Tat-ADJ group
According to this point that speci c neutralizing antibodies are effective in preventing of HPV infection (Wu, Ma et al. 2019), in this study the sera from each vaccinated group (E7-TAT-ADJ, E7-ADJ, E7, E7-TAT, PBS, ADJ) treated with 10 µg/ml of the HPV E7-Tat and HPV E7 protein for examination of speci c IgG antibody production.Analysis of speci c IgG response were assessed against the HPV E7-Tat and HPV E7 protein in various groups in the second and third immunization.Present data shows that antibody response against antigens is high in all vaccinated groups compared to control groups.Indeed, our data indicate that the highest speci c IgG was observed in E7-TAT-ADJ groups among all groups after the second and third immunization (<0.0001).Brie y, our nding shows that the mice immunized with E7-Tat-ADJ protein was able to stimulate strong IgG antibody with compared to other groups (<0.0001).We suggest that Adjuvant in combination with Tat peptide can be effective at elevating the serum antibody level (Fig 7).

3.4
The 100 µg of cell lysate is the safe concentration for toxicity MTT was performed for measuring of toxicity effect of HPV16 and HPV6 cell lysates at different concentration.MTT assay shows that HPV16 and HPV6 cell lysates at ratio of 100 µg did not induce toxic effects compared to 20µg and 50µg during 48 h period.It is interesting that there is no statistical difference was detected in toxicity rate between these groups (Fig 8).

IFN-γ production in E7-TAT-ADJ vaccinated groups
As know, IFN-γ plays a critical role in enhancing the innate and adaptive immune responses against tumor growth.Data indicates that vaccination is an important mechanism for IFN-γ producing.In this present study we examined the protective IFN-γ response in vaccination groups.The splenocyte of all vaccinated groups were stimulated with HPV16, HPV18, HPV6, HPV11 cell lysates, E7-Tat and E7 protein in-vitro.All groups of mice immunization with different vaccine modalities increased the level of IFN-γ secretion as compared to control groups (p < 0.05).The cytokine results shows that the level of IFN-γ secretion in E7-TAT and E7-TAT-ADJ vaccinated groups were effectively increased in compared to other groups (P<0.05)(Fig 9).Brie y, Among all groups the group immunized with the E7-TAT-ADJ showed a signi cant IFN-γ response compared to other groups(P<0.05)(Fig 9).
On the other hand, the splenocyte of all vaccinated groups were stimulated with HPV16, HPV18, HPV6, HPV11 cell lysates, HPV E7-Tat and HPV E7 protein and did not show IL-4 producing in any group (P>0.05,data not shown).

E7-TAT-ADJ induced anti-tumor T cell response
Study shows that expressing and IFN-γ producing CD8+ T cell are important immune response against TC-1 tumors (Kim, Kim et al. 2004); so we examined whether immunization with the novel vaccine could induce E7-speci c cytotoxic activity.In the present study, two weeks after the third immunization, the splenocytes were harvested and the splenocytes of immunized animal were stimulated with E7-Tat, E7 protein and HPV16, HPV18, HPV6, HPV11 cell lysates.As shown in Fig 10, the frequency of CD107a expression in E7-TAT-ADJ vaccinated group was signi cantly higher than in the other groups.Our nding shows that the CTL response in the E7-TAT-ADJ immunized mice is highly effective and enhanced the antigen-speci c immune response.

Therapeutic immunization of tumor-bearing mice with E7-Tat-ADJ and E7-TAT promote overall survival
In the therapeutic strategy, the mice were immunized with E7-Tat, E7-Tat-ADJ, E7, E7-ADJ, PBS and Adjuvant for analysis of protection against tumor development.The results indicated that anti-tumor immune responses elicited by E7-Tat-ADJ and E7-TAT (Fig. 11).
For determination of survival, tumor-bearing mice were monitored 45 days.Fig. 12 shows that 100%(5/5) of animals treated with PBS and ADJ were moribund by day 35.Our results indicate that survival of TC-1bearing mice treated with E7-Tat-ADJ and E7-TAT is signi cantly longer than other groups 60% (3/5) over 45 day period and it could protect mice from tumor growth compared to E7 and E7-ADJ groups.more ever, in the result of current study, 100(5/5) of treated groups with E7 and E7-ADJ were moribund by day 40-41.Our nding suggest that it was statistically signi cant differences between all vaccinated groups (P < 0.0001).

Discussion
In the current study of therapeutic vaccines, the HPV16,18,6,18,6,11 E7 recombinant fusion protein expressed in E.coli with the highest growth and protein synthesis rate.HPVs types encode two non-structural E6 and E7 oncoproteins which induce E6/E7-speci c T cells.E6 protein inactivate tumor suppressor protein P53 and E7 protein bind to pRb which is responsible for the maintenance of the cell cycle (Su, Xu et al. 2016).Our ndings indicate that the recombinant fusion protein is able to induce cellular immune response and CTL activity.Similar to the present study, a review reported that DNA vaccination (VGX-3100) using E7 and E6 protein is able to improve cellular and humoral immune responses, IFN-γ production and CD8 + T cell activation in patients with HPV 16/18-positive CIN2/3 in a phase 1 clinical study (15).
It is well known that APCs capture exogenous antigens through phagocytosis and different mechanisms.The exogenous antigens can be presented on MHC class II.The processing of exogenous antigens by cytosolic pathway can be present by MHC-I and induce the production of speci c CD8 + T cells (Mellman and Steinman 2001).There are several strategies to increase antigen cross-presentation pathways, including the use of gp96 (Singh-Jasuja, Toes et al. 2000), fusionic (Laus, Graddis et al. 2000), membrane protein A of Klebsiella pneumoniae (Jeannin, Renno et al. 2000), and cationic molecules.Tat fusion protein is safe and it is capable of binding antigens to the MHC-I and increases the production of antigen-speci c CD8 + T cells; therefore it is used in cancer therapy, immunotherapy and therapeutic vaccines (Cafaro, Tripiciano et al. 2019).This present study indicate that the Tat peptide of HIV-1 is essential for potentiating the induction of cell-mediated immunity.A result of study demonstrated that cationic proteins such as HIV-Tat sequences, is able to plays an important role for transferring proteins and drugs (20-200KD) to the mammalian cells (Tanaka, Dowdy et al. 2003).Another nding suggested that, protein vaccination conjugated Tat protein is able to improve CTL, IFN-γ and NK cells.Also it has been reported that E7 (49-57) Tat (49-57) nanoparticle plays an effective role to improve CD8 + T cellmediated antitumor immunity (Zhao, Wang et al. 2013).
Freund's adjuvants are known as a delivery system and can be used to enhance the anti-tumor cellular immune responses.These Adjuvants are used in protein vaccines and able to increase the production of innate cytokines such as IFN-γ, IL-12, IL-18, and IL-15 which are activated NK cell and CD107a expression (Mousavi, Saravi et al. 2019).Freund's adjuvants alone are not insu cient for CD8 + T cell priming and tumor regression.In the present research, it was demonstrated that Freund's adjuvants with TAT peptide in E7-TAT-ADJ vaccinated groups are potent adjuvants in priming of immune response and antitumor activity and also increase the survival of tumor-bearing mice.It has been shown that vaccination with HPV-16 cVLP containing CFA (Freund's complete adjuvant) and IFA (Freund's incomplete adjuvant) enhances the production of IgG antibodies and suppresses tumor growth by production of tumor-speci c CD4 + T cells (Monroy-García, Gómez-Lim et al. 2014).A research study indicated that immunization with L1, E6 and E7 protein plus CFA and IFA adjuvant is able to increases the prophylactic e cacy (Xu, Wang et al. 2016).Furthermore another ndings suggested that vaccination with E7 and L1 peptide combined with IFA adjuvant elicits a strong humoral and cellular immunity and increases the CD8 + T cell anti-tumor response and anti-HPV antibodies (Bates, Uematsu et al. 2009) (Levy, Goriely et al. 2013).
Three types of HPV vaccines including Gardasil, Cervarix and Gardasil 9 based on L1 protein are prophylactic and can induce long-term immune responses.Because of the importance of cervical cancer in community, many research have focused on various therapeutic vaccines.This present results showed that HPV16,18,6,11 E7-Tat recombinant fusion protein with Freund's adjuvant is a novel vaccine which is able to increase CTL response and improve speci c antibody in animal model.This vaccine let to increase survival rate by improving CD8 + T cell; so it should be appreciate vaccine against HPV.

Declarations
Funding: This study was funded by Mazandaran University of Medical Sciences, Iran with grant No. 3049 and conducted in the Molecular and Cell Biology Research Center, Faculty of Medicine.
Con ict of interest: Author that no con ict of interest.
Ethical approval: All procedures performed in studies involving mice participants were in accordance with the ethical guideline of the International Council for Laboratory Animal Science (ICLAS) ( IR. MAZUMS.REC.96.3049).This article does not contain any studies with human participants performed by any of the authors.