Abstract
The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.
Highlights
Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, relies on acquiring iron from the host iron-binding glycoproteins, transferrin (Tf) or lactoferrin (Lf), for survival in the human genitourinary tract [1, 2]
The β-strands flanking the four loops on the transferrin binding protein B (TbpB) C-lobe that were not resolved in the crystal structure were selected as the sites for insertion of foreign epitopes
The lobes of the TbpB and Lf binding protein B (LbpB) lipoproteins are comprised of two adjacent anti-parallel β-barrel like structures
Summary
The causative agent of the sexually transmitted infection gonorrhea, relies on acquiring iron from the host iron-binding glycoproteins, transferrin (Tf) or lactoferrin (Lf), for survival in the human genitourinary tract [1, 2] This process is mediated by surface receptor proteins initially discovered in the related human pathogen Neisseria meningitidis [3, 4] and are presumed to be essential for survival of N. meningitidis both in the human upper respiratory tract during asymptomatic colonization and during invasive infection. The bacterial Tf and Lf receptor systems are each composed of an anchored lipoprotein, Tf or Lf binding protein B (TbpB, LbpB) that extends away from the bacterial surface to bind ironloaded Tf or Lf, and the integral membrane proteins Tf or Lf binding protein A (TbpA, LbpA) that transfer iron across the outer membrane These receptors are each exquisitely specific for human Tf and Lf [5], extending detectable binding activity only to apes but not monkeys [6]. It is noteworthy to recognize that the TbpA-Tf interaction is present in pathogens of multiple important food production animals, including poultry, swine, and cattle, [8, 9], suggesting that the host specificity has evolved over a period of more than 300 million years, when the Synapsids (mammalian lineage) split from Sauropsids (bird, reptile lineage)
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