Azam Bolhassani

In Silico Design and Immunological Studies of Two Novel Multiepitope DNA-Based Vaccine Candidates Against High-Risk Human Papillomaviruses

Human papillomaviruses (HPV)-16 and 18 are the most prevalent types associated with cervical cancer. HPV L1 and L2 capsid proteins and E7 oncoprotein play crucial roles in HPV-related diseases. Hence, these proteins were proposed as target antigens for preventive and therapeutic vaccines. In this study, two multiepitope DNA-based HPV vaccine candidates were designed using in silico analysis including the immunogenic and conserved epitopes of HPV16/18 L1, L2 and E7 proteins (the L1-L2-E7 fusion DNA), and of heat shock protein 70 (HSP70) linked to the L1-L2-E7 DNA construct (the HSP70-L1-L2-E7 fusion DNA). Next, the expression of the L1-L2-E7 and HSP70-L1-L2-E7 multiepitope DNA constructs was evaluated in a mammalian cell line. Finally, immunological responses and antitumor effects of the DNA constructs were investigated in C57BL/6 mice. Our data indicated high expression rates of the designed multiepitope L1-L2-E7 DNA (~ 56.16%) and HSP70-L1-L2-E7 DNA (~ 80.45%) constructs in vitro. The linkage of HSP70 epitopes to the L1-L2-E7 DNA construct significantly increased the gene expression. Moreover, the HSP70-L1-L2-E7 DNA construct could significantly increase immune responses toward Th1 response and CTL activity, and induce stronger antitumor effects in mouse model. Thus, the designed HSP70-L1-L2-E7 DNA construct represents promising results for development of HPV DNA vaccine candidates.

Synergistic effects of exosomal crocin or curcumin compounds and HPV L1-E7 polypeptide vaccine construct on tumor eradication in C57BL/6 mouse model

Cervical cancer is the most common malignant tumor in females worldwide. Human papillomavirus (HPV) infection is associated with the occurrence of cervical cancer. Thus, developing an effective and low-cost vaccine against HPV infection, especially in developing countries is an important issue. In this study, a novel HPV L1-E7 fusion multiepitope construct designed by immunoinformatics tools was expressed in bacterial system. HEK-293T cells-derived exosomes were generated and characterized to use as a carrier for crocin and curcumin compounds. The exosomes loaded with crocin and curcumin compounds as a chemotherapeutic agent (ExoCrocin and ExoCurcumin) were used along with the L1-E7 polypeptide for evaluation of immunological and anti-tumor effects in C57BL/6 mouse model.In vitrostudies showed that ExoCrocin and ExoCurcumin were not cytotoxic at a certain dose, and they could enter tumor cells.In vivostudies indicated that combination of the L1-E7 polypeptide with ExoCrocin or ExoCurcumin could produce a significant level of immunity directed toward Th1 response and CTL activity. These regimens showed the protective and therapeutic effects against tumor cells (the percentage of tumor-free mice: ~100%). In addition, both ExoCrocin and ExoCurcumin represented similar immunological and anti-tumor effects. Generally, the use of exosomal crocin or curcumin forms along with the L1-E7 polypeptide could significantly induce T-cell immune responses and eradicate tumor cells.

Current and future direction in treatment of HPV-related cervical disease

Human papillomavirus (HPV) is the most common sexually transmitted virus in the world. About 70% of cervical cancers are caused by the most oncogenic HPV genotypes of 16 and 18. Since available prophylactic vaccines do not induce immunity in those with established HPV infections, the development of therapeutic HPV vaccines using E6 and E7 oncogenes, or both as the target antigens remains essential. Also, knocking out the E6 and E7 oncogenes in host genome by genome-editing CRISPR/Cas system can result in tumor growth suppression. These methods have shown promising results in both preclinical and clinical trials and can be used for controlling the progression of HPV-related cervical diseases. This comprehensive review will detail the current treatment of HPV-related cervical precancerous and cancerous diseases. We also reviewed the future direction of treatment including different kinds of therapeutic methods and vaccines, genome-editing CRISPR/Cas system being studied in clinical trials. Although the progress in the development of therapeutic HPV vaccine has been slow, encouraging results from recent trials showed vaccine-induced regression in high-grade CIN lesions. CRISPR/Cas genome-editing system is also a promising strategy for HPV cancer therapy. However, its safety and specificity need to be optimized before it is used in clinical setting.