Adjuvant-free pH-controlled aggregates of E. coli-expressed H1N1-RBD enhance neutralizing antibody responses and confer protection against influenza virus.

In this study, we used the E. coli-expressed recombinant influenza H1N1 receptor-binding domain (H1N1-RBD; MW = 27.4 kDa) as a model to explore how stable oligomers or aggregates can be produced by modulating sample conditions (temperature and pH), and to examine whether such colloidal/conformational modulations enhance immunogenicity toward vaccine antigen design. We thus analyzed the biophysical properties of the protein under three different pHs at 25 °C and 37 °C using DLS, SLS, CD, and tryptophan fluorescence spectroscopy, and analyzed the immune response in mice. We found that the E. coli-expressed H1N1-RBD was predominantly monomeric at pH 4.7, oligomerized at pH 6.0 (Rh ∼ 20 nm), and formed large aggregates (Rh < 1000 nm) at pH 7.4. Immunization studies in Jcl:ICR and BALB/c mice revealed that the oligomeric form (pH 6.0) elicited the highest IgG titers, whereas the monomeric (pH 4.7) and the aggregated (pH 7.4) forms induced weaker antibody responses. Despite this, all three preparations generated neutralizing antisera, with neutralization potency increasing in the order oligomers < monomers < aggregates, in the absence of adjuvants. Notably, the aggregated H1N1-RBD conferred protection against live viral challenge comparable to that of an inactivated influenza vaccine, as demonstrated by plaque reduction assays in MDCK cells and in vivo challenge experiments in BALB/c mice. Flow cytometric analysis further revealed robust long-term immune memory, with central memory CD4+ T cells accounting for 52.53-65.04% and CD8+ T cells for 23.81-37.85% of the population. Overall, our findings highlight the potential of E. coli-produced proteins as vaccine antigens and suggest that pH-controlled protein oligomerization/aggregation can significantly boost immunogenicity and the neutralizing efficiency of antisera.