Infect Immun. 2015 Mar;83(3):1030-8.

Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

 

Frandoloso R1, Martínez-Martínez S2, Calmettes C3, Fegan J4, Costa E4, Curran D4, Yu RH4, Gutiérrez-Martín CB2, Rodríguez-Ferri EF2, Moraes TF3, Schryvers AB5.
  • 1Laboratory of Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, São José, Passo Fundo, Brazil Department of Animal Health, University of León, León, Spain.
  • 2Department of Animal Health, University of León, León, Spain.
  • 3Department of Biochemistry, University of Toronto, Toronto, Canada.
  • 4Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, Calgary, Canada.
  • 5Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, Calgary, Canada schryver@ucalgary.ca.

Abstract

Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

PMID: 25547790

 

Supplement:

Summary Statement:

This study demonstrates that elimination of the binding activity of transferrin binding protein B produces a dramatically superior vaccine antigen, arguing for renewed efforts at developing improved human and veterinary vaccines against the important pathogens that rely on these surface receptors for survival and disease causation.

 

Background:

A common adaptation among several important Gram-negative bacterial pathogens of humans and food production animals is that they possess surface receptors that specifically bind the iron-binding protein, transferrin (Tf) (1) (Table 1). Notably, the receptors are very specific for Tf from the host, thus human pathogens bind human Tf and pig pathogens bind pig Tf (2). The receptor normally consists of a surface lipoprotein, Tf binding protein B (TbpB), and an integral outer membrane protein, Tf binding protein A (TbpA). The role of TbpB is to capture the iron-loaded form of Tf and transfer it to TbpA, which then extracts iron from Tf at the cell surface (Figure 1). The iron is then transported into the cell and either stored or used as a co-factor for enzymes or other redox proteins. The ability of these bacteria to acquire iron independently from neighboring microbes enables them to proliferate on the mucosal surface and sustain growth during invasive infection.

Table 1. Gram-negative bacterial pathogens of humans and food production animals.

tab1

 

 

fig1

Figure 1. Model for capture and iron removal from Tf by the bacterial transferrin receptor. Iron-loaded Tf is captured by the surface lipoprotein TbpB, which initiates interaction with TbpA via the anchoring peptide, resulting in binding of Tf by TbpA and release of iron.

Their accessibility at the cell surface and critical role in survival and disease causation (3, 4) make the Tf receptors ideal vaccine targets, and were the subject of fairly extensive investigation soon after their initial discovery over 25 years ago (5). Encouraging results in animal studies with the TbpB protein from Neisseria meningitidis made it a lead candidate for a group B meningococcal vaccine, with the prospect that a broadly cross-protective vaccine might be achieved using 2-3 representative TbpB proteins (6). Unfortunately, disappointing results from a Phase I clinical trial in humans led to abandoning the effort at developing a TbpB-based meningococcal vaccine. Efforts at developing TbpB-based vaccines in food production animals involved direct testing of the vaccine antigens in the host species (pigs, cattle), resulting in their inclusion in some vaccine products. This study was designed to use a surrogate host-pathogen system to address the issue of the poor performance of the TbpB-based meningococcal vaccine that contrasted the prior experimental results from immunization of mice and rabbits.

 

fig2

 

The Study:

One possible explanation for the poor performance of the TbpB-based antigens during Phase I trials in humans was that binding of human Tf to TbpB was interfering with the development of an optimal immune response, which would suggest that there would be an opportunity to develop improved vaccines against both human animal pathogens. To address this issue we prepared a non-binding mutant of TbpB from the porcine pathogen Haemophilus parasuis that was nearly identical in structure to the wild-type protein and assessed its ability to prevent infection. The results (Figure 2) demonstrated that the mutant protein provided complete protection against infection, whereas only 3/6 pigs immunized with the wild-type protein survived, and these had substantial symptoms and pathology from the infection (Figure 2). This provides strong evidence that Tf was interfering with induction of an optimal protective immune response in the natural host (pigs). It is noteworthy to mention that immunization with the mutant TbpB also provided substantially superior protection than a commercial vaccine product in this experiment (Figure 2). The commercial vaccine was meant to serve as a positive control as it had provided protection from infection in prior experiments (7). The failure of the commercial vaccine to protect against infection in this experiment and the clinical characteristics of the infection indicates that there was an effectively higher dose of virulent bacteria in the challenge preparation. This highlights the degree to which the mutant TbpB induced a superior protective immune response against infection.

fig3

 

Since the bacteria were introduced by intra-tracheal injection it is uncertain whether the enhanced protective immune response was due to actions on the mucosal surfaces of the lower respiratory tract and/or during invasive stages of the infection. However, since the mutant and wild-type proteins were virtually identical except for the absence of an amino acid side chain, it seems likely that the enhanced protective response was due to antibodies directed against the surface of TbpB involved in binding Tf (Figure 3), promoting different immune or biological effects on the bacteria. One mechanism that might contribute to the enhanced protection is the inhibition of binding of Tf and subsequent uptake of iron, thus limiting the growth of the bacteria. This effect may be more important on the mucosal surface where TbpB has been shown to be important for survival (4), likely due to more limiting levels of Tf since the TbpB-dependence was not readily demonstrated under laboratory conditions that are more reflective of Tf levels within the body. Testing whether the mutant TbpB provides greater protection against colonization under natural conditions would not only address this question, but would provide additional reasons for developing Tf receptor-based vaccines. Vaccines that can affect colonization rather than simply prevent infection have the added advantage of impacting the transmission of the pathogens to non-immunized individuals (herd immunity).

Overall this study suggests that substantially improved vaccines targeting the Tf receptors can be developed against important human and veterinary pathogens through protein engineering, and provides additional support for the view that these receptors are ideal vaccine targets.

 

 

References:

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  7. Frandoloso R, Martínez S, Rodríguez-Ferri EF, García-Iglesias MJ, Pérez-Martínez C, Martinez-Fernandez B, Gutierrez-Martin CB. 2011. Development and characterization of protective Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin and comparison with other subunit and commercial vaccines. Clinical and Vaccine Immunology 18:50-58.

 

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