Generic placeholder image

Current Pharmaceutical Biotechnology


ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Research Article

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

Author(s): Manica Negahdaripour, Navid Nezafat, Reza Heidari, Nasrollah Erfani, Nasim Hajighahramani, Mohammad B. Ghoshoon, Eskandar Shoolian, Mohammad R. Rahbar, Sohrab Najafipour, Ali Dehshahri, Mohammad H. Morowvat and Younes Ghasemi*

Volume 21, Issue 4, 2020

Page: [316 - 324] Pages: 9

DOI: 10.2174/1389201020666191114104850

Price: $65


Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches.

Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines.

Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group.

Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

Keywords: Human Papillomavirus (HPV), L2, cervical cancer, epitope vaccine, flagellin, TLR4 agonist, TLR5 agonist, adjuvant.

Graphical Abstract
Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer, 2015, 136(5), E359-E386.
[] [PMID: 25220842]
Wang, J.W.; Hung, C.F.; Huh, W.K.; Trimble, C.L.; Roden, R.B. Immunoprevention of human papillomavirus-associated malignancies. Cancer Prev. Res. (Phila.), 2015, 8(2), 95-104.
[] [PMID: 25488410]
Pouyanfard, S.; Müller, M. Human papillomavirus first and second generation vaccines-current status and future directions. Biol. Chem., 2017, 398(8), 871-889.
[] [PMID: 28328521]
Schiller, J.T.; Lowy, D.R. Raising expectations for subunit vaccine. J. Infect. Dis., 2015, 211(9), 1373-1375.
[] [PMID: 25420478]
Negahdaripour, M.; Eslami, M.; Nezafat, N.; Hajighahramani, N.; Ghoshoon, M.B.; Shoolian, E.; Dehshahri, A.; Erfani, N.; Morowvat, M.H.; Ghasemi, Y. A novel HPV prophylactic peptide vaccine, designed by immunoinformatics and structural vaccinology approaches. Infect. Genet. Evol., 2017, 54, 402-416.
[] [PMID: 28780192]
Kalnin, K.; Tibbitts, T.; Yan, Y.; Stegalkina, S.; Shen, L.; Costa, V.; Sabharwal, R.; Anderson, S.F.; Day, P.M.; Christensen, N.; Schiller, J.T.; Jagu, S.; Roden, R.B.; Almond, J.; Kleanthous, H. Low doses of flagellin-L2 multimer vaccines protect against challenge with diverse papillomavirus genotypes. Vaccine, 2014, 32(28), 3540-3547.
[] [PMID: 24780250]
Negahdaripour, M.; Golkar, N.; Hajighahramani, N.; Kianpour, S.; Nezafat, N.; Ghasemi, Y. Harnessing self-assembled peptide nanoparticles in epitope vaccine design. Biotechnol. Adv., 2017, 35(5), 575-596.
[] [PMID: 28522213]
Wu, W.H.; Alkutkar, T.; Karanam, B.; Roden, R.B.; Ketner, G.; Ibeanu, O.A. Capsid display of a conserved human papillomavirus L2 peptide in the adenovirus 5 hexon protein: A candidate prophylactic hpv vaccine approach. Virol. J., 2015, 12(140), 140.
[] [PMID: 26362430]
Lee, S.E.; Hong, S.H.; Verma, V. Flagellin is a strong vaginal adjuvant of a therapeutic vaccine for genital cancer. OncoImmunology, 2016, 5(2), e1081328-e1.
Reed, S.G.; Hsu, F.C.; Carter, D.; Orr, M.T. The science of vaccine adjuvants: Advances in TLR4 ligand adjuvants. Curr. Opin. Immunol., 2016, 41, 85-90.
[] [PMID: 27392183]
Vijay-Kumar, M.; Gewirtz, A.T. Flagellin: Key target of mucosal innate immunity. Mucosal Immunol., 2009, 2(3), 197-205.
[] [PMID: 19242410]
Song, L.; Zhang, Y.; Yun, N.E.; Poussard, A.L.; Smith, J.N.; Smith, J.K.; Borisevich, V.; Linde, J.J.; Zacks, M.A.; Li, H.; Kavita, U.; Reiserova, L.; Liu, X.; Dumuren, K.; Balasubramanian, B.; Weaver, B.; Parent, J.; Umlauf, S.; Liu, G.; Huleatt, J.; Tussey, L.; Paessler, S. Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin. Vaccine, 2009, 27(42), 5875-5884.
[] [PMID: 19654064]
Nguyen, C.T.; Hong, S.H.; Sin, J.I.; Vu, H.V.; Jeong, K.; Cho, K.O.; Uematsu, S.; Akira, S.; Lee, S.E.; Rhee, J.H. Flagellin enhances tumor-specific CD8+T cell immune responses through TLR5 stimulation in a therapeutic cancer vaccine model. Vaccine, 2013, 31(37), 3879-3887.
[] [PMID: 23831323]
Leng, J.; Stout-Delgado, H.W.; Kavita, U.; Jacobs, A.; Tang, J.; Du, W.; Tussey, L.; Goldstein, D.R. Efficacy of a vaccine that links viral epitopes to flagellin in protecting aged mice from influenza viral infection. Vaccine, 2011, 29(45), 8147-8155.
[] [PMID: 21854824]
Taylor, D.N.; Treanor, J.J.; Sheldon, E.A.; Johnson, C.; Umlauf, S.; Song, L.; Kavita, U.; Liu, G.; Tussey, L.; Ozer, K.; Hofstaetter, T.; Shaw, A. Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin fusion vaccine with improved safety and immune response. Vaccine, 2012, 30(39), 5761-5769.
[] [PMID: 22796139]
Aflatoonian, R.; Fazeli, A. Toll-like receptors in female reproductive tract and their menstrual cycle dependent expression. J. Reprod. Immunol., 2008, 77(1), 7-13.
[] [PMID: 17493683]
Hajam, I.A.; Dar, P.A.; Shahnawaz, I.; Jaume, J.C.; Lee, J.H. Bacterial flagellin-a potent immunomodulatory agent. Exp. Mol. Med., 2017, 49(9), e373
[] [PMID: 28860663]
Shanmugam, A.; Rajoria, S.; George, A.L.; Mittelman, A.; Suriano, R.; Tiwari, R.K. Synthetic Toll like receptor-4 (TLR-4) agonist peptides as a novel class of adjuvants. PLoS One, 2012, 7(2), e30839
[] [PMID: 22363498]
Zhu, Q.; Egelston, C.; Vivekanandhan, A.; Uematsu, S.; Akira, S.; Klinman, D.M.; Belyakov, I.M.; Berzofsky, J.A. Toll-like receptor ligands synergize through distinct dendritic cell pathways to induce T cell responses: implications for vaccines. Proc. Natl. Acad. Sci. USA, 2008, 105(42), 16260-16265.
[] [PMID: 18845682]
Mäkelä, S.M.; Strengell, M.; Pietilä, T.E.; Osterlund, P.; Julkunen, I. Multiple signaling pathways contribute to synergistic TLR ligand-dependent cytokine gene expression in human monocyte-derived macrophages and dendritic cells. J. Leukoc. Biol., 2009, 85(4), 664-672.
[] [PMID: 19164128]
Orr, M.T.; Beebe, E.A.; Hudson, T.E.; Moon, J.J.; Fox, C.B.; Reed, S.G.; Coler, R.N. A dual TLR agonist adjuvant enhances the immunogenicity and protective efficacy of the tuberculosis vaccine antigen ID93. PLoS One, 2014, 9(1), e83884
[] [PMID: 24404140]
Weir, G.M.; Karkada, M.; Hoskin, D.; Stanford, M.M.; MacDonald, L.; Mansour, M.; Liwski, R.S. Combination of poly I:C and Pam3CSK4 enhances activation of B cells in vitro and boosts antibody responses to protein vaccines in vivo. PLoS One, 2017, 12(6), e0180073
[] [PMID: 28662082]
Zhu, Q.; Egelston, C.; Gagnon, S.; Sui, Y.; Belyakov, I.M.; Klinman, D.M.; Berzofsky, J.A. Using 3 TLR ligands as a combination adjuvant induces qualitative changes in T cell responses needed for antiviral protection in mice. J. Clin. Invest., 2010, 120(2), 607-616.
[] [PMID: 20101095]
Napolitani, G.; Rinaldi, A.; Bertoni, F. Selected TLR agonist combinations synergistically trigger a TH1 polarizing program in dendritic cells. Nat. Immunol., 2005, 6(8), 769-776.
[] [PMID: 15995707]
Atapour, A.; Negahdaripour, M.; Ghasemi, Y. n silico designing a candidate vaccine against breast cancer. Int. J. Pep. Res. Ther., 2019, 1-12.
Dorosti, H.; Eslami, M.; Negahdaripour, M.; Ghoshoon, M.B.; Gholami, A.; Heidari, R.; Dehshahri, A.; Erfani, N.; Nezafat, N.; Ghasemi, Y. Vaccinomics approach for developing multi-epitope peptide pneumococcal vaccine. J. Biomol. Struct. Dyn., 2019, 37(13), 3524-3535.
[] [PMID: 30634893]
Hajighahramani, N.; Eslami, M.; Negahdaripour, M.; Ghoshoon, M.B.; Dehshahri, A.; Erfani, N.; Heidari, R.; Gholami, A.; Nezafat, N.; Ghasemi, Y. Computational design of a chimeric epitope-based vaccine to protect against Staphylococcus aureus infections. Mol. Cell. Probes, 2019, 46, 101-414.
[] [PMID: 31233779]
Vakili, B.; Nezafat, N.; Hatam, G.R.; Zare, B.; Erfani, N.; Ghasemi, Y. Proteome-scale identification of Leishmania infantum for novel vaccine candidates: A hierarchical subtractive approach. Comput. Biol. Chem., 2018, 72, 16-25.
[] [PMID: 29291591]
Negahdaripour, M.; Nezafat, N.; Hajighahramani, N.; Rahmatabadi, S.S.; Ghasemi, Y. Investigating CRISPR-Cas systems in Clostridium botulinum via bioinformatics tools. Infect. Genet. Evol., 2017, 54, 355-373.
[] [PMID: 28684374]
Rahmatabadi, S.S.; Sadeghian, I.; Nezafat, N. In silico investigation of pullulanase enzymes from various bacillus species. Curr. Proteomics, 2017, 14, 175-185.
Negahdaripour, M.; Nezafat, N.; Hajighahramani, N. In silico study of different signal peptides for secretory production of interleukin-11 in Escherichia coli. Curr. Proteomics, 2017, 14(1), 112-121.
Doorbar, J.; Egawa, N.; Griffin, H.; Kranjec, C.; Murakami, I. Human papillomavirus molecular biology and disease association. Rev. Med. Virol., 2015, 25(Suppl. 1), 2-23.
[] [PMID: 25752814]
Alphs, H.H.; Gambhira, R.; Karanam, B.; Roberts, J.N.; Jagu, S.; Schiller, J.T.; Zeng, W.; Jackson, D.C.; Roden, R.B. Protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of L2. Proc. Natl. Acad. Sci. USA, 2008, 105(15), 5850-5855.
[] [PMID: 18413606]
Tyler, M.; Tumban, E.; Dziduszko, A.; Ozbun, M.A.; Peabody, D.S.; Chackerian, B. Immunization with a consensus epitope from human papillomavirus L2 induces antibodies that are broadly neutralizing. Vaccine, 2014, 32(34), 4267-4274.
[] [PMID: 24962748]
Zhai, L.; Peabody, J.; Pang, Y.Y.S. A novel candidate MS2 phage VLP vaccine displaying a tandem HPV L2 peptide offers similar protection in mice to Gardasil-9. Antiviral Res., 2017, 147, 116-123.
[] [PMID: 28939477]
Negahdaripour, M.; Nezafat, N.; Eslami, M.; Ghoshoon, M.B.; Shoolian, E.; Najafipour, S.; Morowvat, M.H.; Dehshahri, A.; Erfani, N.; Ghasemi, Y. Structural vaccinology considerations for in silico designing of a multi-epitope vaccine. Infect. Genet. Evol., 2018, 58, 96-109.
[] [PMID: 29253673]
Karimi, Z.; Nezafat, N.; Negahdaripour, M.; Berenjian, A.; Hemmati, S.; Ghasemi, Y. The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli. Protein Expr. Purif., 2015, 114, 108-114.
[] [PMID: 26118697]
Ghasemi, Y.; Hajighahramani, N.; Dabbagh, F.; Ghoshoon, M.B.; Yarahmadi, E.; Mobasher, M.A. Cloning, expression, purification and expression condition optimization of α-enolase from Staphylococcus aureus in Escherichia coli. Minerva Biotecnol., 2016, 28(1), 33-38.
Ghoshoon, M.B.; Berenjian, A.; Hemmati, S. Extracellular production of recombinant l-asparaginase II in Escherichia coli: Medium optimization using response surface methodology. Int. J. Pept. Res. Ther., 2015, 21(4), 487-495.
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685.
[] [PMID: 5432063]
Nezafat, N.; Sadraeian, M.; Rahbar, M.R.; Khoshnoud, M.J.; Mohkam, M.; Gholami, A.; Banihashemi, M.; Ghasemi, Y. Production of a novel multi-epitope peptide vaccine for cancer immunotherapy in TC-1 tumor-bearing mice. Biologicals, 2015, 43(1), 11-17.
[] [PMID: 25467837]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72(1–2), 248-254.
[] [PMID: 942051]
Pinto, L.A.; Castle, P.E.; Roden, R.B.; Harro, C.D.; Lowy, D.R.; Schiller, J.T.; Wallace, D.; Williams, M.; Kopp, W.; Frazer, I.H.; Berzofsky, J.A.; Hildesheim, A. HPV-16 L1 VLP vaccine elicits a broad-spectrum of cytokine responses in whole blood. Vaccine, 2005, 23(27), 3555-3564.
[] [PMID: 15855014]
Brewer, J.M.; Conacher, M.; Satoskar, A.; Bluethmann, H.; Alexander, J. In interleukin-4-deficient mice, alum not only generates T helper 1 responses equivalent to freund’s complete adjuvant, but continues to induce T helper 2 cytokine production. Eur. J. Immunol., 1996, 26(9), 2062-2066.
Hogarth, P.M.; Pietersz, G.A. Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond. Nat. Rev. Drug Discov., 2012, 11(4), 311-331.
[] [PMID: 22460124]
Bahls, L.; Yamakawa, R.; Zanão, K.; Alfieri, D.; Flauzino, T.; Delongui, F.; de Abreu, A.; Souza, R.; Gimenes, F.; Reiche, E.; Borelli, S.; Consolaro, M. Human Leukocyte antigen class I and class II polymorphisms and serum cytokine profiles in cervical cancer. Int. J. Mol. Sci., 2017, 18(9), 1-15.
[] [PMID: 28858203]
Strestik, B.D.; Olbrich, A.R.M.; Hasenkrug, K.J.; Dittmer, U. The role of IL-5, IL-6 and IL-10 in primary and vaccine-primed immune responses to infection with Friend retrovirus (Murine leukaemia virus). J. Gen. Virol., 2001, 82(Pt 6), 1349-1354.
Turner, M.D.; Nedjai, B.; Hurst, T.; Pennington, D.J. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim. Biophys. Acta, 2014, 1843(11), 2563-2582.
[] [PMID: 24892271]
Day, P.M.; Thompson, C.D.; Lowy, D.R.; Schiller, J.T. interferon gamma prevents infectious entry of human Papillomavirus 16 via an L2-dependent mechanism. J. Virol., 2017, 91(10), e00168-e17.
[] [PMID: 28250129]
García-Piñeres, A.; Hildesheim, A.; Dodd, L.; Kemp, T.J.; Williams, M.; Harro, C.; Lowy, D.R.; Schiller, J.T.; Pinto, L.A. Cytokine and chemokine profiles following vaccination with human papillomavirus type 16 L1 Virus-like particles. Clin. Vaccine Immunol., 2007, 14(8), 984-989.
[] [PMID: 17596432]
Herrin, D.M.; Coates, E.E.; Costner, P.J.; Kemp, T.J.; Nason, M.C.; Saharia, K.K.; Pan, Y.; Sarwar, U.N.; Holman, L.; Yamshchikov, G.; Koup, R.A.; Pang, Y.Y.; Seder, R.A.; Schiller, J.T.; Graham, B.S.; Pinto, L.A.; Ledgerwood, J.E. Comparison of adaptive and innate immune responses induced by licensed vaccines for human papillomavirus. Hum. Vaccin. Immunother., 2014, 10(12), 3446-3454.
[] [PMID: 25483691]
Dupuy, C.; Buzoni-Gatel, D.; Touze, A.; Le Cann, P.; Bout, D.; Coursaget, P. Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles. Microb. Pathog., 1997, 22(4), 219-225.
[] [PMID: 9140917]
Tsukui, T.; Hildesheim, A.; Schiffman, M.H.; Lucci, J., III; Contois, D.; Lawler, P.; Rush, B.B.; Lorincz, A.T.; Corrigan, A.; Burk, R.D.; Qu, W.; Marshall, M.A.; Mann, D.; Carrington, M.; Clerici, M.; Shearer, G.M.; Carbone, D.P.; Scott, D.R.; Houghten, R.A.; Berzofsky, J.A. Interleukin 2 production in vitro by peripheral lymphocytes in response to human papillomavirus-derived peptides: correlation with cervical pathology. Cancer Res., 1996, 56(17), 3967-3974.
[PMID: 8752165]
Emeny, R.T.; Wheeler, C.M.; Jansen, K.U.; Hunt, W.C.; Fu, T.M.; Smith, J.F.; MacMullen, S.; Esser, M.T.; Paliard, X. Priming of human papillomavirus type 11-specific humoral and cellular immune responses in college-aged women with a virus-like particle vaccine. J. Virol., 2002, 76(15), 7832-7842.
[] [PMID: 12097595]
Evans, T.G.G.; Bonnez, W.; Rose, R.C.C.; Koenig, S.; Demeter, L.; Suzich, J.A.; O’Brien, D.; Campbell, M.; White, W.I.; Balsley, J.; Reichman, R.C. A Phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers. J. Infect. Dis., 2001, 183(10), 1485-1493.
[] [PMID: 11319684]
Wang, Y.; Liu, X.H.; Li, Y.H.; Li, O. The paradox of IL-10-mediated modulation in cervical cancer. Biomed. Rep., 2013, 1(3), 347-351.
[] [PMID: 24648946]
Berti, F.C.B.; Pereira, A.P.L.; Cebinelli, G.C.M.; Trugilo, K.P.; Brajão de Oliveira, K. The role of interleukin 10 in human papilloma virus infection and progression to cervical carcinoma. Cytokine Growth Factor Rev., 2017, 34, 1-13.
[] [PMID: 28365229]
Nakayama, T.; Kashiwagi, Y.; Kawashima, H. Long-term regulation of local cytokine production following immunization in mice. Microbiol. Immunol., 2018, 62(2), 124-131.
[] [PMID: 29266448]
Shibaki, A.; Katz, S.I. Induction of skewed Th1/Th2 T-cell differentiation via subcutaneous immunization with Freund’s adjuvant. Exp. Dermatol., 2002, 11(2), 126-134.
[] [PMID: 11994139]
Chuang, Y.H.; Chiang, B.L.; Chou, C.C.; Hsieh, K.H. Immune effector cells induced by complete Freund’s adjuvant exert an inhibitory effect on antigen-specific type 2 T helper responses. Clin. Exp. Allergy, 1997, 27(3), 315-324.
[] [PMID: 9088658]
Paradkar, P.H.; Joshi, J.V.; Mertia, P.N.; Agashe, S.V.; Vaidya, R.A. Role of cytokines in genesis, progression and prognosis of cervical cancer. Asian Pac. J. Cancer Prev., 2014, 15(9), 3851-3864.
[] [PMID: 24935564]
Wei, L.H.; Kuo, M.L.; Chen, C.A.; Chou, C.H.; Lai, K.B.; Lee, C.N.; Hsieh, C.Y. Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway. Oncogene, 2003, 22(10), 1517-1527.
[] [PMID: 12629515]
Brewer, J.M.; Conacher, M.; Gaffney, M.; Douglas, M.; Bluethmann, H.; Alexander, J. Neither interleukin-6 nor signalling via tumour necrosis factor receptor-1 contribute to the adjuvant activity of Alum and Freund’s adjuvant. Immunology, 1998, 93(1), 41-48.
[] [PMID: 9536117]
Pinto, L.A.; Edwards, J.; Castle, P.E.; Harro, C.D.; Lowy, D.R.; Schiller, J.T.; Wallace, D.; Kopp, W.; Adelsberger, J.W.; Baseler, M.W.; Berzofsky, J.A.; Hildesheim, A. Cellular immune responses to Human Papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles. J. Infect. Dis., 2003, 188(2), 327-338.
[] [PMID: 12854090]
Gonçalves, A.K.; Giraldo, P.C.; Machado, P.R.; Farias, K.J.; Costa, A.P.; Freitas, J.C.; Eleutério, J., Jr; Witkin, S.S. Human papillomavirus vaccine-induced cytokine messenger RNA expression in vaccinated women. Viral Immunol., 2015, 28(6), 339-342.
[] [PMID: 25965646]
Jiao, X.D.; Cheng, S.; Hu, Y.H.; Sun, L. Comparative study of the effects of aluminum adjuvants and Freund’s incomplete adjuvant on the immune response to an Edwardsiella tarda major antigen. Vaccine, 2010, 28(7), 1832-1837.
[] [PMID: 20006568]
Littleton, L.C.; Gruenke, J.A. A comparison of the effect of three adjuvants on the antibody response to ovalbumin in mice. BIOS, 2013, 84(3), 142-147.

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy