Multi-Component Vaccine Candidates Against Non-Typeable Haemophilus influenzae

<b>Background:</b><i>Haemophilus influenzae</i> (Hi), a Gram-negative bacterium, is divided into two broad categories: encapsulated and non-capsulated isolates, also called non-typeable Hi isolates (NTHi). NTHi has become prevalent since the introduction of the vaccine against Hi of serotype b. Hi can cause local infections on respiratory mucosal surfaces and urogenital infections, which can lead to septic abortion in pregnant women. It can also cause invasive infections such as meningitis and septicemia. Moreover, NTHi isolates are becoming increasingly resistant to antibiotics. Vaccines targeting NTHi are not yet available. As these NTHi isolates are not encapsulated, vaccines should target proteins at the bacterial surface. However, vaccine development is hindered by the high variability of these proteins. We aimed to identify conserved outer membrane proteins (OMPs) for vaccines against NTHi. <b>Methods:</b> We analyzed core-genome multilocus sequence typing (cgMLST) of 1144 genomes of Hi collected between 2017 and 2022 and, of these, identified 514 conserved genes that encoded OMPs. We focused on two specific OMPs: Haem1295, encoding the protein P5 (P5), and Haem1040, encoding the protein 26 (P26). P5 is known to bind human complement regulatory protein factor H (FH), while both P5 and P26 are involved in enhancing immune responses. The genes encoding these proteins were cloned, overexpressed, purified, and tested in both active and passive protection models using systemic infection in mice. <b>Results:</b> P5 and P26 were found to be immunogenic during human infections. Vaccination with these proteins conferred protection against both homologous and heterologous NTHi isolates in mice, suggesting broad cross-protection. <b>Conclusions:</b> P5 and P26 are promising vaccine candidates showing cross-protection against NTHi and offering the additional benefit of targeting bacterial virulence factors, enhancing vaccine efficacy against NTHi isolates.