Linear
DNA vaccines provide effective vaccination. However, their application is limited by high cost and small scale of the conventional polymerase chain reaction (PCR) generally used to obtain sufficient amounts of
DNA effective against epidemic diseases. In this study, a two-step, large-scale PCR was established using a low-cost
DNA polymerase, RKOD, expressed in Pichia pastoris. Two linear
DNA vaccines encoding
influenza H1N1
hemagglutinin (HA) 1, LEC-HA, and PTO-LEC-HA (with phosphorothioate-modified primers), were produced by the two-step PCR. Protective effects of the
vaccines were evaluated in a mouse model. BALB/c mice were immunized three times with the
vaccines or a control
DNA fragment. All immunized animals were challenged by
intranasal administration of a lethal dose of
influenza H1N1 virus 2 weeks after the last immunization. Sera of the immunized animals were tested for the presence of HA-specific
antibodies, and the total IFN-γ responses induced by linear
DNA vaccines were measured. The results showed that the
DNA vaccines but not the control
DNA induced strong antibody and IFN-γ responses. Additionally, the PTO-LEC-HA
vaccine effectively protected the mice against the lethal homologous mouse-adapted virus, with a survival rate of 100% versus 70% in the LEC-HA-vaccinated group, showing that the PTO-LEC-HA
vaccine was more effective than LEC-HA. In conclusion, the results indicated that the linear H1N1 HA-coding
DNA vaccines induced significant immune responses and protected mice against a lethal virus challenge. Thus, the low-cost, two-step, large-scale PCR can be considered a potential tool for rapid manufacturing of linear
DNA vaccines against
emerging infectious diseases.