Immunogenesity Enhancement of Influenza Virus Stalk Domain Using Leishmania Major Heat Shock Protein, One Step Closer to Universal Vaccine

Influenza viruses cause acute respiratory infections in humans, which can often be accompanied by pandemics with varying rates of illness and even a fetal end. Current vaccines target the variable globular part of hemagglutinin (HA); so, their effectiveness is limited and they need perpetual updating. To prepare universal vaccine, scientist's effort is focused on conserved antigenic parts of influenza virus such as hemagglutinin stalk domain which is more conserved compare to its globular head. The aim of this study was evaluating the immunogenicity of influenza A (H1N1) HA2 peptide in mouse model. To prepare HA2 protein, the expression vector encoding the gene of interest (PET-28a/HA2), was transformed into Escherichia coli BL21-DE3 competent cells. Recombinant HA2 protein was extracted and purified using Ni-TED columns under denaturing conditions, refolded and desalted via step-wise dialysis. The HA2 concentration was determined via using Bradford protein assay. Six-week-old BALB/c mice in different groups were immunized intradermally with HA2 alone or supplemented with Freund or Lm.HSP70221-604 as adjuvants. Two groups were injected with HSP70 and phosphate buffered saline (PBS) as negative controls. Humeral immunity in vaccinated mice was measured using the enzyme-linked immunosorbent assay (ELISA); and finally, mice were challenged with one lethal dose (LD90) of PR8 virus. The protein expression, extraction and purification stages were confirmed using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis. The highest antibody response was measured in mice vaccinated with HA2-Freund, as expected. Specific antibody response in group received HA2-HSP70 was of higher titer than HA2 alone. More over HA2-HSP70 group surprisingly showed higher level of protection against lethal H1N1 (PR8) challenge compared to other groups. It has been proved that HSP70 can induce specific response and innate immunity via stimulating CD8+ T-Cells, CD40, TLR2, TLR4 and cytokine secretion. Our results showed that HA2 alone or supplemented with HSP70 protected mice against lethal influenza challenge and could be considered as a universal vaccine.