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 ... -  《-》

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 ... -  《-》

Development of HCV Therapeutic Vaccines Using Hp91 Peptide and Small Heat Shock Protein 20 as an Adjuvant.

Hepatitis C Virus (HCV) is a major cause of chronic liver disease in the world. Many studies showed that T-cell responses to HCV Nonstructural Protein 3 (NS3) play an important role in clearing acute HCV infections, thus NS3 can be considered as a suitable candidate antigen for development of a HCV therapeutic vaccine. We used Hp91 peptide and small heat shock protein 20 as an adjuvant for enhancement of NS3-specific humoral and cellular immunity in mice. In this study, various NS3 DNA and protein constructs were generated in eukaryotic and prokaryotic expression systems, and their potency in eliciting humoral and cellular immune responses was compared using small Heat shock protein 20 (Hsp20), the High Mobility Group Box 1 protein (HMGB1)-derived peptide (Hp91), and Freund's adjuvant in a BALB/c mouse model. Our results indicated that both the Hsp20 conjugated with NS3 protein and Hp91 significantly enhanced the levels of IgG2a, IgG2b and IFN-γ directed toward Th1 responses compared to other groups. Moreover, the immunostimulatory properties of Hp91 were significantly higher than Hsp20 and Freund's adjuvant in various immunization strategies. Mice immunized by NS3 protein formulated with Freund's adjuvant, and also mixed with Hp91 peptide showed higher secretion of Granzyme B than other groups. Hp91 peptide could develop NS3-specific B- and T-cell immune responses as a promising HCV therapeutic vaccine candidate in future.

Basirnejad M ，Bolhassani A 《-》

Significance of serum antibodies against HPV E7, Hsp27, Hsp20 and Hp91 in Iranian HPV-exposed women.

Ramezani A ，Aghakhani A ，Soleymani S ，Bavand A ，Bolhassani A ... -  《BMC INFECTIOUS DISEASES》

Immunomodulatory effects of IP-10 chemokine along with PEI600-Tat delivery system in DNA vaccination against HPV infections.

Although DNA vaccines represent an attractive approach for generating antigen-specific immunity, improvement of their potency is highly demanded. In the present study, three strategies including linkage to immunostimulatory molecules (N-terminal of gp96), co-administration of chemokines (IP-10 or RANTES) and PEI600-Tat as non-viral gene delivery system have been applied to enhance DNA vaccine efficacy against HPV infections. We found that C57BL/6 immunization with E7-NT-gp96 fusion gene led to increased level of IFN-γ compared to E7 alone. The fused genes showed considerable protective potency in tumor mice model. In addition, E7-NT-gp96 delivered with PEI600-Tat was more protective against E7-expressing tumors comparing with E7-NT-gp96 alone. Our results showed that co-administration of IP-10 with E7-NT-gp96 delivered by PEI600-Tat elicits significant IFN-γ production and consequently a strong preventive response against TC-1 tumor cells in contrast to increased tumor growth by RANTES co-delivery. Also in therapeutic experiment, our data showed that co-immunization of IP-10 at the same inoculation site of TC-1 along with E7-NT-gp96 delivery by PEI600-Tat is able to significantly suppress TC-1 tumor growth. The successful treatment by this immunization protocol was associated with the elevated levels of IFN-γ and IL-2 production in the lymph nodes. These data indicated that fusion of NT-gp96 to E7 in combination with IP-10 co-administration and PEI600-Tat delivery system can synergistically enhance the potency of HPV DNA vaccines. Therefore, this approach suggests a combinational therapeutic strategy against cervical and other HPV-related cancers.

Mohit E ，Bolhassani A ，Zahedifard F ，Seyed N ，Eslamifar A ，Taghikhani M ，Samimi-Rad K ，Rafati S ... -  《-》