Nontypeable Haemophilus influenzae (NTHi) is responsible for diseases such as otitis media, sinusitis, bronchitis, and pneumonia. Unlike H. influenzae Serotype b (Hib), there is no vaccine against diseases induced by NTHi. High molecular weight1 (HMW1), high molecular weight 2 (HMW2), and H. influenzae type a (Hia) proteins are critical protein adhesions of NTHi with the potentiality to provide the protection. Objectives: The current study aimed at investigating the potential of recombinant

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... 555-914, HMW2553-916, and Hia585-705 proteins as subunit vaccines to induce immune responses after active immunization in Bagg albino (inbred research mouse strain) or Balb/c mice. Methods: HMW1555-914, HMW2553-916, and Hia585-705 amplified by polymerase chain reaction (PCR) were cloned into pET28a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blotting analysis were carried out to investigate the characteristics of proteins. Proteins were purified by Ni-nitrilotriacetic acid (Ni-NTA) as a gel matrix. Mice (Balb/c) were immunized subcutaneously with HMW1555-914, HMW2553-916, and Hia585-705 proteins, alone or in binary and ternary combinations. Eventually, specific antibody responses were evaluated by the enzyme-linked immunosorbent assay (ELISA) and serum bactericidal assay (SBA). Results: Antibody responses significantly increased in Balb/c immunized by ternary combination compared with the control group. IgG1/IgG2a ratio indicated that proteins directed immune responses toward T-helper 2. Antisera produced against purified proteins in ternary combination showed the highest bactericidal activity against NTHi strains with the titer of 1:32. Conclusions: The obtained results suggested that HMW1555-914, HMW2553-916, and Hia585-705 adhesins were the potential subunit vaccine candidates of NTHi and after further investigation could be considered for protection against NTHi infections.