Monoclonal Antibodies against Vibrio vulnificus RtxA1 Elicit Protective Immunity through Distinct Mechanisms

Abstract

g Vibrio vulnificus causes rapidly progressing septicemia with an extremely high mortality rate (>50%), even with aggressive antibiotic treatment. The bacteria secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins, which are involved in the pathogenesis of Gram-negative Vibrio species. Recently, we reported that immunization with the C-terminal region of V. vulnificus RtxA1/MARTX Vv , RtxA1-C, elicits a protective immune response against V. vulnificus through a poorly defined mechanism. In this study, we generated a panel of new monoclonal antibodies (MAbs) against V. vulnificus RtxA1-C and investigated their protective efficacies and mechanisms in a mouse model of infection. Prophylactic administration of seven MAbs strongly protected mice against lethal V. vulnificus infection (more than 90% survival). Moreover, three of these MAbs (21RA, 24RA, and 47RA) demonstrated marked efficacy as postexposure therapy. Notably, 21RA was therapeutically effective against lethal V. vulnificus infection by a variety of routes. Using Fab fragments and a neutropenic mouse model, we showed that 21RA and 24RA mediate protection from V. vulnificus infection through an Fc-independent and/or neutrophil-independent pathway. In contrast, 47RA-mediated protection was dependent on its Fc region and was reduced to 50% in neutropenic mice compared with 21RA-mediated and 24RA-mediated protection. Bacteriological study indicated that 21RA appears to enhance the clearance of V. vulnificus from the blood. Overall, these studies suggest that humoral immunity controls V. vulnificus infection through at least two different mechanisms. Furthermore, our panel of MAbs could provide attractive candidates for the further development of immunoprophylaxis/therapeutics and other therapies against V. vulnificus that target the MARTX toxin.V ibrio vulnificus, a member of the normal marine microbiota, is an important human pathogen associated with the consumption of seafood and wound infection following exposure to water containing this pathogen (1-4). The bacterial infection frequently progresses to severe skin lesions and septicemia in people with hepatic disorders or immunocompromised conditions (5). Despite support care and aggressive antibiotic therapy, V. vulnificus septicemia has a greater than 50% mortality rate; this rate increases to more than 90% for patients with septic shock (5-8). During the past decade, the incidence of V. vulnificus infection has increased worldwide, probably due to the warming of coastal waters (9, 10).V. vulnificus produces a wide range of potential virulence factors required for survival and growth, including capsular polysaccharide (VvPS), iron assimilation systems, flagella, pili, VvhA, VvpE, and the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin (MARTX Vv , or RtxA1) (8, 11). Among them, V. vulnificus RtxA1/MARTX Vv , a large secreted protein, belongs to the repeats-in-toxin (RTX) toxin family, which has been identified in a number of Gram-negative bacterial pathogens (12, 13). V. vuln...

Development and characterization of a catalytically inactive cysteine protease domain of RtxA1/MARTXVv as a potential vaccine for Vibrio vulnificus

“…The identity of this protein was confirmed by western blotting with an anti‐RtxA1‐C monoclonal antibody (22RA) and anti‐serum from V. vulnificus M06‐24/O‐infected mice (Fig. c and data not shown) ; equivalent amounts (100 ng) of purified GST (Fig. c, lane 1) and CPD Vv (C3725S) (Fig.…”

Cross‐protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv

“…Recent studies have shown that protective immunity against Vibrio species is induced by both bacterial components and toxins, including LPS, pili, capsular polysaccharides, cholera toxins and MARTX toxins . Among these protective immunogens, MARTX toxins, which comprise a subfamily of large secreted RTX proteins, have been identified in a number of Vibrio species and other pathogenic gram‐negative bacteria .…”

“…We previously generated a panel of mAbs against the C‐terminal region (aa residues 3491–4701) of V. vulnificus RtxA1/MARTX Vv , RtxA1‐C . We also characterized the protective activity of anti‐RtxA1‐C mAbs in mouse models of V. vulnificus infection .…”

“…We previously generated a panel of mAbs against the C‐terminal region (aa residues 3491–4701) of V. vulnificus RtxA1/MARTX Vv , RtxA1‐C . We also characterized the protective activity of anti‐RtxA1‐C mAbs in mouse models of V. vulnificus infection . Although an earlier study reported cross‐protection between different V. vulnificus strains , no mAbs against V. vulnificus RtxA1/MARTX Vv have yet been investigated for their ability to inhibit other Vibrio species.…”

“…We also characterized the protective activity of anti‐RtxA1‐C mAbs in mouse models of V. vulnificus infection . Although an earlier study reported cross‐protection between different V. vulnificus strains , no mAbs against V. vulnificus RtxA1/MARTX Vv have yet been investigated for their ability to inhibit other Vibrio species. To evaluate whether mAbs against V. vulnificus confer protective activity against different Vibrio species with MARTX toxins, we first assessed their immunoreactivity by ELISA using MARTX Vc proteins from the V. cholerae O1 biotype El Tor strain N16961 because there are highly conserved domains between the C‐terminal region of V. vulnificus RtxA1/MARTX Vv and V. cholerae MARTX Vc proteins (Fig.…”