Combination of built-in “TLR۴/۵ agonists/tetanus toxoid and TLR۷ agonist/alum adjuvants elicits robust Th۱/Th۲ anti-HPV L۲ RG-۱ responses in immunized mice

BACKGROUND AND ABJECTIVE Human papillomavirus (HPV), a heterogeneous group of around ۲۰۰ types that infect the epithelia of the skin and mucosa in humans, is the most common sexual infection and the main cause of cervical cancer. Currently available HPV vaccines (Cervarix and Gardasil/۹) are highly efficient in the prevention of infection due to induction of type-specific and high titer neutralizing antibody (nAb) against major capsid protein (L۱) of the virus. Although these vaccines protect from major cancer producing HPV types such as ۱۶, ۱۸, ۳۱ and ۴۵, but type-specificity (limited cross-protections), high cost and technical complexity of the production process, restrict their versatile utilization especially in developing countries. Several prior studies indicated that HPV minor capsid protein (L۲) contains a conserved pan-type linear so called RG۱ epitope (amino acids ۱۷-۳۶) capable of eliciting broadly cross-reactive nAbs against different HPV types, albeit in much lower titers and potency compared to the L۱-based VLP. To this end, several strategies such as multiplication of the RG۱ epitope and using various adjuvants were employed to enhance its immunogenicity.MATERIALS AND METHODSHerein, we designed a construct harboring the dual ۳x tandem repeats of the HPV۱۶ RG۱ epitope (TP), the Entolimod (the TLR۵ agonist), the short peptide “RS۰۹ (the TLR۴ agonist)” and the tetanus toxoid P۲ epitope (TT-P۲) which were linked by the (GGGS)۳ linker in tandem, and evaluated for immunogenicity in BALB/c mice with different adjuvant formulations.RESULTS AND DISCUSSION Analyses by SDS-PAGE and Western blot indicated that the expression of this construct in E.coli produced the expected ۴۶ kDa protein (hereafter; rejoined peptide (RP). Groups of mice were immunized (either subcutaneously (SC) or intramuscularly (IM) by RP alone and TP or RP formulated with different adjuvant formulations containing TLR۷ agonists (in the form of either injectable R۸۳۷ imiquimod or Aldara imiquimod cream (AL) for skin treatment) and Alum adjuvants. Assessment of the humoral responses and IFN-γ secretion as well as cellular proliferation showed that mice immunized by RP-alone or RP-containing formulations (RP+Alum+R۸۳۷, RP+Alum, RP+R۸۳۷, RP) induced significantly higher Nabs and higher IFN-γ secretion and cellular proliferation compared to those immunized by any TP-containing formulations (TP+Alum+R۸۳۷, TP+Alum, TP+R۸۳۷, TP). ۲۰۶These results indicated the pivotal role of the designed built-in adjuvant within the RP immunogen for induction of anti-RG۱ responses. Moreover, the highest level of humoral and cellular responses (IFN-γ secretion and cellular proliferation) was reached in mice immunized SC by “RP+TLR۷ agonist/alum adjuvants” formulations (RP+Alum+R۸۳۷ and RP+Alum+AL) compared to other RP-immunized mice groups. This observation indicated that TLR۷ agonists (R۸۳۷ or Aldara) /alum adjuvants synergistically enhanced the crucial effect of built-in adjuvants on the induction of anti-HPV RG۱ immune responses. Moreover, assessment of IgG۱/IgG۲a ratios indicated that groups receiving TLR۷ agonists (R۸۳۷ or Aldara) by SC immunization (RP+Alum+R۸۳۷, RP+Alum+AL or RP+R۸۳۷) showed a balanced Th۱/Th۲ polarization of the immune system, while groups deprived of TLR۷ agonists or received TLR۷ (R۸۳۷) via IM immunization route showed a Th۲ phenotype.CONCLUSION Collectively, our data showed the synergistic effect of TLR۷ agonist/alum to enhance the crucial role of the built-in “TLR۴/۵ agonists / (TT)-P۲” for induction of robust and balanced Th۱/Th۲ anti-HPV RG۱ immune responses. Results of this study might help to achieve a promising vaccine formulation for the induction of anti-HPV L۲ RG۱ immune responses for production of an inexpensive pan-genomic and cross-reactive HPV vaccine.

کلیدواژه ها:

HPV ، RG۱ epitope ، Built-in adjuvants ، Tetanus toxoid ، Neutralizing antibody