The association between anti-PcrV titers and protective effects against P. aeruginosa in PcrV vaccinated mice Saeko Hamaoka1, Yoshifumi Naito1, Hideya Kato1, Atsushi Kainuma1, Teiji Sawa1: 1Kyoto Prefectural University of Medicine, Kyoto, Japan INTRODUCTION On day 60, the PcrV-specific IgG titers of the immunized mice were measured with ELISA. On day 65, the immunized mice were infected intratracheally with a lethal dose of PA103, a cytotoxic strain of P. aeruginosa. The survival was monitored over 24 h, then the lungs from each mouse were collected and homogenized to measure bacterial counts, lung edema indexes and myeloperoxidase (MPO) activity levels in the lungs. (a) (b) Pseudomonas aeruginosa is one of the most common pathogens which cause acute pneumonia in critically ill or immunocompromised patients, and treatment of P. aeruginosa infections has become difficult because of resistance to multiple antibiotics (1). Development of a vaccine against P. aeruginosa is a potential strategy to prevent infections and improve the poor prognosis, but no licensed vaccine is clinically available (2). The Kaplan–Meier estimator and a log-rank test were used to assess survival. One-way Analysis of Variance and Bonferroni test were used to compare anti-PcrV titers. Canonical correlation analysis was used to evaluate correlation between anti-PcrV titers and infection-related values. A p value of less than 0.05 was considered statistically significant. Figure 2. (a) Anti-PcrV titers in the sera of vaccinated mice. (b) Survival rates in the vaccinated mice at 24 hr after the infection with P. aeruginosa. *P<0.01 vs. saline; †P<0.05 vs PcrV alone. We have focused on PcrV as a vaccine antigen, which is the cap structure of the type III secretion system (TTSS), a major virulence mechanism of P. aeruginosa (Fig. 1). Blocking PcrV can impede the translocation of secretory toxins (ExoS, ExoT, ExoU and ExoY) into the host cells through the TTSS, and as a result improve the lethal outcome of P. aeruginosa pneumonia. ExoU ExoT ExoS ExoY P. aeruginosa Host cell Outer membrane Cytoplasmic membrane Peptidoglycan layer PcrV Type III secretion system (a) (b) Figure 3. Correlation between anti-PcrV titers and (a) survival rates, (b) bacterial counts in the lungs, (c) lung edema indexes, and (d) MPO activity levels at 24 hr after the infection with P. aeruginosa. Group Antigen (dose/mouse) Adjuvant (dose/mouse) PcrV-FA rePcrV (10µg) Freund’s adjuvant (100µl) PcrV-alum alum (100µl) PcrV-CpG CpG ODN (10µg) PcrV alone none alum alone CpG alone saline (c) (d) Figure 1. Type III secretion system.. OBJECTIVES Table 1. Mouse vaccination groups. rePcrV: recombinant PcrV, alum: aluminum hydroxide, CpG ODN: CpG oligodeoxynucleotide. . Vaccination with recombinant PcrV improved the mortality of mice infected with virulent P. aeruginosa (3,4). However, the vaccine contained Freund’s adjuvant (FA), which cannot be applied to humans. In order to develop an effective human vaccine against P. aeruginosa, we combined PcrV with a clinically applicable adjuvant, aluminum hydroxide (alum) or CpG oligodeoxynucleotide (CpG ODN), and examined the efficacies in a mouse model of P. aeruginosa pneumonia, from the viewpoint of inducing PcrV-specific immunity. RESULTS CONCLUSIONS After the vaccinations, the PcrV-FA, PcrV-alum and PcrV-CpG groups had significantly higher anti-PcrV titers than the saline group (*p < 0.01) and the PcrV alone group († p < 0.05) (Fig. 2a). At 24 h after the infection, the survival rates of the PcrV-FA, PcrV-alum and PcrV-CpG groups were 91%, 73%, 64%, respectively, all of which significantly improved compared with that of the saline group (*p < 0.01, Fig. 2b). The survival rates of the immunized groups were significantly positively correlated with the anti-PcrV titers (R2=0.9758, p<0.001, Fig. 3a). In addition, there was a significant negative correlation between the anti-PcrV titers and the bacterial counts (R2=0.7502, p<0.05, Fig. 3b), the lung edema indexes (R2=0.7796, p<0.01, Fig. 3c), and the MPO activity levels (R2=0.6389, p<0.05, Fig. 3d) at 24 h post-infection. This study shows that the enhanced anti-PcrV titers induced by PcrV-FA, PcrV-alum and PcrV-CpG are closely related to the improved survival rates, the decreased bacterial counts, the reduced levels of lung edema and MPO activity after the infection with P. aeruginosa pneumonia. PcrV-alum and PcrV-CpG could be promising human vaccine candidates against P. aeruginosa. METHODS Male ICR mice (4 weeks old) were used, and the protocols for all animal experiments were approved in advance by the Animal Research Committee of the institute. On day 0, 20 and 40, mice (n=11 per group) were immunized intraperitoneally with one of the vaccines or solutions (Table 1). REFERENCES 1. Chest 139: 1172-85. 2011 2. Expert Rev Vaccines 13: 507–19. 2014 3. Nat Med 5: 392–8. 1999 4. Microbiol Immunol 53: 587–94. 2009