Immuno-Stimulating Peptide Derived from HMGB1 is More Effective Than the N-Terminal Domain of Gp96 as an Endogenous Adjuvant for Improvement of Protein Vaccines.

Up to now, different protein vaccine modalities against human papillomavirus (HPV) have been designed to control cervical cancer. The important issue is to increase their immunogenicity using appropriate adjuvants. Among heat shock proteins (HSPs), glycoprotein 96 (Gp96) and its Nterminal region (NT-gp96) have attracted a specific interest in stimulation of antigen-specific immune responses in vivo. Furthermore, the potency of high mobility group box 1 (HMGB1) protein and its fragment (Hp91) was reported to enhance the desired immune responses against various disorders. In this study, the recombinant (r) HPV16 E7 and rNT-gp96 proteins were generated in bacterial expression system. Mice were vaccinated three times with E7 antigen mixed with Montanide, Hp91, and NT-gp96 as the adjuvant and their preventive and therapeutic efficiencies were evaluated in a murine tumor model. Mice vaccinated with E7 co-delivered by Hp91 peptide induced higher IgG2a and IFN-γ responses in comparison with E7 co-injected with Montanide and NT-gp96 protein suggesting a strong Th1 cellular immune response. The data showed that vaccination with noncovalent rE7 + rNT-gp96 complex delayed the tumor growth as compared to control groups. Mice immunized with rE7 + Montanide and rE7 + Hp91 protected 100% of mice versus 75% survival in groups vaccinated with rE7 + rNT-gp96 after TC-1 tumor challenge. The percentage of tumor free mice was decreased in group immunized with rE7 + rNT-gp96 in therapeutic experiments (~ 50%). These results demonstrated that Hp91 peptide is a safe and strong adjuvant against rNT-gp96 with the potent anti-tumor effects similar to Montanide adjuvant.

Talebi S ，Bolhassani A ，Azad TM ，Arashkia A ，Modaresi MH ... -  《-》

The contribution of NT-gp96 as an adjuvant for increasing HPV16 E7-specific immunity in C57BL /6 mouse model.

To control cervical cancer, efficient vaccination against human papillomavirus (HPV) is highly required. Despite the advantages and safety of the protein vaccines, additional strategies to enhance their immunogenicity are needed. E7 is a transforming protein which represents a perfect target antigen for vaccines or immunotherapies. Heat shock proteins (HSPs) facilitate cellular immune responses to antigenic peptides or proteins bound to them. Regarding to previous studies, vaccination with purified HSP/antigen complexes efficiently elicit antigen-specific immune responses in mice model. The N-terminal of glycoprotein 96 (NT-gp96) has adjuvant effect and can induce effective cumulative immune response against clinical disorders, especially cancers. In this study, the recombinant HPV16 E7 and E7 linked to NT-gp96 (E7-NT-gp96) proteins were generated in prokaryotic expression system. Mice were vaccinated twice with this recombinant proteins and the immunogenicity of the fusion protein was determined. The preventive efficacy of E7-NT-gp96 fusion protein was also evaluated and compared to E7 protein after challenging with cancerous TC-1 cell line. In vitro re-stimulated splenocytes of mice vaccinated with rE7-NT-gp96 protein induced higher IFN-γ response in comparison with E7 protein immunization. Moreover, immunization with E7-NT-gp96 protein displayed low but stable humoral responses at post-challenge time. The data showed that vaccination with fused E7-NT-gp96 protein delayed the tumour occurrence and growth as compared to protein E7 alone. These results suggest that fused adjuvant-free E7-NT-gp96 protein vaccination could direct the immune responses towards Th1 immunity. Furthermore, the linkage of NT-gp96 to E7 could enhance protective anti-tumour immunity.

Mohit E ，Bolhassani A ，Zahedifard F ，Taslimi Y ，Rafati S ... -  《-》

Enhanced immunogenicity of HPV16E7 accompanied by Gp96 as an adjuvant in two vaccination strategies.

Bolhassani A ，Zahedifard F ，Taghikhani M ，Rafati S ... -  《VACCINE》

Different domains of glycoprotein 96 influence HPV16 E7 DNA vaccine potency via electroporation mediated delivery in tumor mice model.

DNA vaccines have emerged as a promising approach for generating antigen-specific immunotherapy. However, due to their low immunogenicity, there is a need to enhance DNA-based vaccine potency. Two main strategies to increase DNA-based vaccine potency are the employment of immuno-adjuvants such as heat shock proteins (HSPs) and a method of improving the delivery of naked plasmid DNA by electroporation. In the current study, we evaluated the effects of linkage of human papillomavirus (HPV) type 16 E7 as a model antigen to N-terminal and C-terminal of glycoprotein 96 (NT-/CT-gp96) on the potency of E7-specific immunity generated by DNA vaccines. We found that subcutaneous DNA injection with E7-CT (gp96) followed by electroporation generates the significant E7-specific IFN-γ immune responses as well as the best protective effects in vaccinated mice as compared to E7 or E7-NT (gp96) DNA vaccines. Therefore, our data indicate that subcutaneous administration of E7 DNA linked to CT (gp96) fragment followed by electroporation can significantly enhance the potency of DNA vaccines. Indeed, the structural domains of immuno-chaperones show the potential of generating effective immune responses against different clinical disorders such as cancer. Altogether, our results show that comparable regions of gp96 (N-/C-terminal fragments of gp96) may have qualitatively different immunological effects in vaccine design.

Daemi A ，Bolhassani A ，Rafati S ，Zahedifard F ，Hosseinzadeh S ，Doustdari F ... -  《-》

Hp91 immunoadjuvant: An HMGB1-derived peptide for development of therapeutic HPV vaccines.

High percentage of human cervical malignancy is related to human papillomavirus (HPV) infections. Thus, it is important to find novel non-invasive treatment strategies among various therapeutic HPV vaccines. In current study, we investigated the protective and therapeutic effects of DNA- and protein-based vaccines using HPV16 E7 as a model antigen in tumor mice model. In this line, the full length of high-mobility group box 1 (HMGB1) protein as well as an HMGB1-derived short peptide (Hp91) was used as an adjuvant for stimulating adaptive immunity and developing the potency of these vaccines. DNA vaccination of HPV16 E7 with HMGB1 was performed as the complexed and conjugated forms. The immunostimulatory properties of Hp91 peptide along with Hp121 control peptide were compared to Montanide 720 in protein vaccination. Our data showed that co-immunization of HPV16 E7 protein with Hp91 peptide or Hp91+Hp121 peptides significantly increased the secretion of IFN-γ, IgG2a antibody response, and protected 100% of mice against a TC-1 tumor challenge. Furthermore, the linkage of HMGB1 with E7 antigen led to enhance the immunogenicity of DNA vaccine especially in combination with Hp91 and Hp121 peptides. These findings suggest that Hp91 peptide, and the full length of HMGB1 gene could be an efficient adjuvant for improvement of therapeutic HPV protein- and DNA-based vaccines, respectively.

Talebi S ，Bolhassani A ，Sadat SM ，Vahabpour R ，Agi E ，Shahbazi S ... -  《-》