Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes fatal acute lung infections in critically ill individuals. Its pathogenesis is associated with bacterial virulence conferred by the type III secretion system (TTSS), through which P. aeruginosa causes necrosis of the lung epithelium and disseminates into the circulation, resulting in bacteremia, sepsis, and mortality. TTSS allows P. aeruginosa to directly translocate cytotoxins into eukaryotic cells, inducing cell death. The P. aeruginosa V-antigen PcrV, a homolog of the Yersinia V-antigen LcrV, is an indispensable contributor to TTS toxin translocation. Vaccination against PcrV ensures the survival of challenged mice and decreases lung inflammation and injury. Both the rabbit polyclonal anti-PcrV antibody and the murine monoclonal anti-PcrV antibody, mAb166, inhibit TTS toxin translocation. mAb166 IgG was cloned, and a molecular engineered humanized anti-PcrV IgG antigen-binding fragment, KB001, was developed for clinical use. KB001 is currently undergoing Phase-II clinical trials for ventilator-associated pneumonia in France and chronic pneumonia in cystic fibrosis in USA. In these studies, KB001 has demonstrated its safety, a favorable pharmacokinetic profile, and promising potential as a nonantibiotic strategy to reduce airway inflammation and damage in P. aeruginosa pneumonia.

Keywords: CF, cystic fibrosis; Fab, fragment antigen binding; Fc, fragment crystallizable region; MDR, multidrug resistant; MDRP, multidrug resistant Pseudomonas aeruginosa; P. aeruginosa, Pseudomonas aeruginosa; PcrV; Pseudomonas aeruginosa; TTS, type III secretory; TTSS, type III secretion system; V-antigen; VAP, ventilator-associated pneumonia; antibody; immunoglobulin G, IgG; mAb, monoclonal antibody; type III secretion system.

Figure 1.

The type III secretory apparatus of Pseudomonas aeruginosa: A basal component, a needle structure, and a translocon. The type III secretory (TTS) apparatus also known as the injectisome comprises many protein components. The basal component comprises an outer ring PscC, periplasmic joint PscJ, ATPase PscN, and more, and is the mechanism by which TTS toxins pass through the bacterial cell membrane. The needle structure comprises approximately 140 PscF proteins. The translocon comprises PcrV, PopB, and PopD, and is the mechanism by which TTS toxins translocate through the eukaryotic cell membrane. PcrV forms a cap structure on the tip of the secretory needle rod.

Figure 2.

The mAb166 blocking epitope of PcrV. PcrV consists of 294 amino acids. The C-terminal center region between amino acids 144–257, consisting of 114 amino acids and located between 2 coiled-coil regions, is the blocking epitope for the monoclonal anti-PcrV IgG antibody, mAb166. This blocking epitope overlaps with both the channel-forming region for erythrocyte hemolysis and the blocking region of the homologous Yersinia LcrV, based on a sequence alignment between PcrV and LcrV. mAb166 binds to this epitope and blocks the translocation of type III secretory toxins. Amino acid 204 corresponds to the hypervariable region of Yersinia LcrV.

Figure 3.

Predicted tertiary structure of PcrV. Based on the structural information for LcrV (Swissplot IR6F), the tertiary structure of PAO1 PcrV was predicted by the protein structure prediction server RaptorX. PcrV, consisting of 294 amino acids, forms a central shaft structure, with coiled-coil double strands and 2 globular domains at either end. The C-terminal globular domain includes amino acid 204, corresponding to the hypervariable region of Yersinia LcrV, and amino acids 144–257, recognized as the mAb166 blocking epitope.

Figure 4.

PcrV forms a ring structure at the tip of the secretion needle. A. Monomeric PcrV. B. Five PcrV aligned in a circle. C. Top view of the ring structure formed by pentameric PcrV.