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

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.

Structural vaccinology considerations for in silico designing of a multi-epitope vaccine.

Multi-epitope peptide vaccines, as a kind of fusion proteins, usually possess a string-of-beads structure, consisting of several peptidic epitopes, probably adjuvants and linkers. Very numerous options are possible in selecting the order of different segments and linkers. Such factors can affect the vaccine efficacy through impacting physicochemical characteristics and protein tertiary structure. To investigate such relations, eleven different constructs were designed and studied as a multi-epitope prophylaxis vaccine for human papilloma virus (HPV). The vaccine contained two epitopes from the minor protein of virus capsid (L2) of HPV16, two TLR agonists as adjuvants (flagellin and RS09, as TLR5 and TLR4 agonists, respectively), and two universal T-helper epitopes. Since the used TLR4 agonist was inserted in the middle of the construct, its appropriate interaction with the bulky TLR4 was a serious concern. Thus, beyond evaluating the physicochemical properties, secondary and tertiary structures, and conformational B-cell epitopes of the designed constructs, TLR4 agonist exposability was also studied. Besides, the interaction between TLR4 and its agonist was investigated through docking and MD studies. Consequently, one structure ("D") with proper physicochemical features, a high frequency of conformational B-cell epitopes, and appropriate interactions with TLR4 and TLR5 in docking and MD studies, was selected as a proper candidate. Accordingly, for in silico designing of multi-epitope vaccines, structural concerns should be considered, and the linkers and arrangement of epitopes and adjuvants should be optimized. Considering the diversity of the possible structures, devising computational tools for such investigations would be very valuable.

A novel HPV prophylactic peptide vaccine, designed by immunoinformatics and structural vaccinology approaches.

Human papillomavirus (HPV)-caused cervical cancer is the fourth common female cancer globally. Despite availability of three effective vaccines in market, development of HPV prophylactic vaccines is still pursued due to affordability issues and type-restricted protection of the marketed vaccines. Investigational second generation prophylactic HPV vaccines are mostly exploiting epitopes from the virus minor capsid protein (L2), which despite many advantages suffer from low immunogenicity, a common problem of epitope vaccines. Adjuvants such as TLR agonists may overcome this drawback. In this study, different immunoinformatics and computational tools were employed to design a novel peptide vaccine for protection against cervical cancer. Two immunodominant epitope domains (amino acids 10-36 and 65-89) from the L2 protein of HPV 16 with potential to promote Th1, Th2, CTL, B-cell, and INF-gamma responses were selected. Flagellin, as a TLR5 agonist, a short synthetic TLR4 agonist, and two universal T-helper agonists (PADRE and TpD) were added to ensure strong induction of immune responses. Different segments were joined by proper linkers, and the physicochemical, structural, and immunological characteristics of the resultant construct were evaluated. Modeling, refinement, and validation were done to achieve a high quality 3D structure of the vaccine protein. Docking and molecular dynamics (MD) studies demonstrated an appropriate and stable interaction between the vaccine and TLR5 during the simulation period. Totally, a potential vaccine candidate with proper immunological and physicochemical properties was designed for HPV prophylaxis. The designed vaccine is expected to be capable of generating humoral and cellular responses, which are vital for protection against HPV.