HPV16 mutant E6/E7 construct is protective in mouse model.

BACKGROUND: Human papillomavirus type 16 (HPV-16) infection is strongly associated with considerable parts of cervical, neck, and head cancers. Performed investigations have had moderate clinical success, so research to reach an efficient vaccine has been of great interest. In the present study, the immunization potential of a newly designed HPV-16 construct was evaluated in a mouse model. RESULTS: Initially, a construct containing HPV-16 mutant (m) E6/E7 fusion gene was designed and antigen produced in two platforms (i.e., DNA vaccine and recombinant protein). Subsequently, the immunogenicity of these platforms was investigated in five mice) C57BL/6 (groups based on several administration strategies. Three mice groups were immunized recombinant protein, DNA vaccine, and a combination of them, and two other groups were negative controls. The peripheral blood mononuclear cells (PBMCs) proliferation, Interleukin-5 (IL-5) and interferon-γ (IFN-γ) cytokines, IgG1 and IgG2a antibody levels were measured. After two weeks, TC-1 tumor cells were injected into all mice groups, and subsequently further analysis of tumor growth and metastasis and mice survival were performed according to the schedule. Overall, the results obtained from in vitro immunology and tumor cells challenging assays indicated the potential of the mE6/E7 construct as an HPV16 therapeutic vaccine candidate. The results demonstrated a significant increase in IFN-γ cytokine (P value < 0.05) in the Protein/Protein (D) and DNA/Protein (E) groups. This finding was in agreement with in vivo assays. Control groups show a 10.5-fold increase (P value < 0.001) and (C) DNA/DNA group shows a 2.5-fold increase (P value < 0.01) in tumor growth compared to D and E groups. Also, a significant increase in survival of D and E (P value < 0.001) and C (P value < 0.01) groups were observed. CONCLUSIONS: So, according to the findings, the recombinant protein could induce stronger protection compared to the DNA vaccine form. Protein/Protein and DNA/Protein are promising administration strategies for presenting this construct to develop an HPV-16 therapeutic vaccine candidate.

Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review.

Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

Determining buffer conditions for downstream processing of VLP-based recombinant hepatitis B surface antigen using multimodal resins in bind-elute and flow-through purification modes.

The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. The purification performance of the resin was constantly retained (97-100%) and no significant resin damage took place after 10 adsorption-elution-cleaning cycles. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast.