Immunization of mice by a multimeric L2-based linear epitope (17-36) from HPV type 16/18 induced cross reactive neutralizing antibodies

Farhad Motavalli Khiavi, Arash Arashkia, Maryam Nasimi, Mehdi Mahdavi, Majid Golkar, Farzin Roohvand, Kayhan Azadmanesh


Current licensed and commercially available prophylactic human papillomavirus (HPV) vaccines (Cervarix and quadrivalent/nine valents Gardasil) are based on major capsid protein L1 virus-like particles (VLPs) production which are expensive and type specific. Minor capsid L2-RG1 linear epitope (17-36) is a known candidate for induction of cross-neutralizing antibodies to develop low-cost pan-HPV vaccines. Herein, we report construction and expression of a three tandem repeats of L2-RG1 derived from HPV16 and 18 fused with the same head to tail pattern (HPV16:17-36×3+ HPV18:17-36×3; hereafter termed dual-type fusion L2 peptide) in E. coli and provide the results of its immunogenicity in mice. SDS-PAGE and western blot analyses indicated proper expression of the peptide that could be further purified by Ni-NTA affinity chromatography via the located C-terminal 6xHis-tag. Mice immunized by formulation of the purified peptide and Freund adjuvant raised neutralizing antibodies which showed proper cross reactivity to HPV L2 (11-200) of types: 18, 16, 31 and 45 (which totally are responsible for 90% of cervical cancers) and efficiently neutralized HPV18/16 pseudoviruses in vitro. Our results imply the possibility of development of a simple, low-cost preventive HPV vaccine based on this dual-type fusion L2 peptide in bacterial expression system with broad spectrum.


Human papilloma virus; Cross neutralization; Pseudovirus and L2 peptide

Full Text:



Doorbar J, Egawa N, Griffin H, Kranjec C, Murakami I. Human papillomavirus molecular biology and disease association. Rev Med Virol. 2015;1:2-23.

Schellenbacher C, Roden RB, Kirnbauer R. Developments in L2-based human papillomavirus (HPV) vaccines. Virus Res. 2017;231:166-175.

Malary M, Moosazadeh M, Hamzehgardeshi Z, Afshari M, Moghaddasifar I, Afsharimoghaddam A. The prevalence of cervical human papillomavirus infection and the most at-risk genotypes among iranian healthy women: a systematic review and meta-analysis. Int J Prev Med. 2016;7:70

Castle PE, Maza M. Prophylactic HPV vaccination: past, present, and future. Epidemiol Infect. 2016;3:449-468.

Pitisuttithum P, Velicer C, Luxembourg A. 9-Valent HPV vaccine for cancers, pre-cancers and genital warts related to HPV. Expert Rev Vaccines. 2015;14(11):1405-19.

Harper DM, Franco EL, Wheeler CM, Moscicki AB, Romonowski B, Roteli-Martins CM, et al. Sustained efficacy up to 4-5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet. 2006 ;367(9518):1247-1255.

Vinodhini K, Shanmughapriya S, Das BC, Natarajaseenivasan K. Prevalence and risk factors of HPV infection among women from various provinces of the world. Arch Gynecol Obstet. 2012;285(3):771-777.

Gambhira R, Jagu S, Karanam B, Gravitt PE, Culp TD, Christensen ND, et al. Protection of rabbits against challenge with rabbit papillomaviruses by immunization with the N terminus of human papillomavirus type 16 minor capsid antigen L2. J Virol. 2007;81(21):11585-11592.

Gambhira R, Karanam B, Jagu S, Roberts JN, Buck CB, Bossis I, et al. A protective and broadly cross-neutralizing epitope of human papillomavirus L2. J Virol. 2007;81(24):13927-13931.

Zhang T, Liu H, Chen X, Wang Z, Wang S, Qu C, et al. Lipidated L2 epitope repeats fused with a single-chain antibody fragment targeting human FcgammaRI elicited cross-neutralizing antibodies against a broad spectrum of human papillomavirus types. Vaccine. 2016;34(46):5531-5539.

Jagu S, Karanam B, Gambhira R, Chivukula SV, Chaganti RJ, Lowy DR, et al. Concatenated multitype L2 fusion proteins as candidate prophylactic pan-human papillomavirus vaccines. J Natl Cancer Inst. 2009; 101(11):782-792.

Rubio I, Bolchi A, Moretto N, Canali E, Gissmann L, Tommasino M, et al. Potent anti-HPV immune responses induced by tandem repeats of the HPV16 L2 (20-38) peptide displayed on bacterial thioredoxin. Vaccine. 2009; 27(13):1949-1956.

Toft L, Tolstrup M, Muller M, Sehr P, Bonde J, Storgaard M, et al. Comparison of the immunogenicity of Cervarix(R) and Gardasil(R) human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults. Hum Vaccin Immunother. 2014;10(5):1147-1154.

Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348(6):518-527.

Sullivan K. Short protocols in molecular biology, 2nd Edn. Trends in Biotechnology. 10:456.

Seitz H, Ribeiro-Muller L, Canali E, Bolchi A, Tommasino M, Ottonello S, et al. Robust in vitro and in vivo neutralization against multiple high-risk hpv types induced by a thermostable thioredoxin-l2 vaccine. Cancer Prev Res. 2015;8(10):932-941.

Day PM, Pang YYS, Kines RC, Thompson CD, Lowy DR, Schiller JT. A human papillomavirus (hpv) in vitro neutralization assay that recapitulates the in vitro process of infection provides a sensitive measure of hpv l2 infection-inhibiting antibodies. Clin Vaccine Immunol. 2012;19(7):1075-1082.

Jagu S, Karanam B, Wang JW, Zayed H, Weghofer M, Brendle SA, et al. Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes. Vaccine. 2015;33(42):5553-5563.

Chen X, Liu H, Zhang T, Liu Y, Xie X, Wang Z, et al. A vaccine of l2 epitope repeats fused with a modified igg1 fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types. PLoS One. 2014;9(5).

Jagu S, Kwak K, Karanam B, Huh WK, Damotharan V, Chivukula SV, et al. Optimization of multimeric human papillomavirus L2 vaccines. PLoS One. 2013;8(1):e55538.

Krajden M, Karunakaran K, So S, Palefsky JM, Sharma R, Cook D, et al. Prevalence of human papillomavirus 16 and 18 neutralizing antibodies in prenatal women in British Columbia. Clin Vaccine Immunol. 2009;16(12):1840-1843.


  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.