Vaccine 32 (2014) 271–276

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Protection against Vibrio vulnificus infection by active and passive immunization with the C-terminal region of the RtxA1/MARTXVv protein Tae Hee Lee a , Mi Hyun Kim a , Chang-Seop Lee b , Ju-Hyung Lee c , Joon Haeng Rhee e,f , Kyung Min Chung a,d,∗ a

Department of Microbiology and Immunology, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-756, Republic of Korea Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-756, Republic of Korea c Department of Preventive Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-756, Republic of Korea d Institute for Medical Science, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-756, Republic of Korea e Clinical Vaccine R&D Center, Chonnam National University Medical School, Gwangju 520-724, Republic of Korea f Department of Microbiology, Chonnam National University Medical School, Gwangju 520-724, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 15 July 2013 Received in revised form 27 September 2013 Accepted 6 November 2013 Available online 17 November 2013 Keywords: Vibrio vulnificus RtxA1/MARTXVv Vaccine Passive immunization Therapy

a b s t r a c t Vibrio vulnificus is a foodborne pathogen that is prevalent in coastal waters worldwide. Infection with V. vulnificus causes septicemia with fatality rates exceeding 50% even with aggressive antibiotic therapy. Several vaccine studies to prevent V. vulnificus infection have been performed but have had limited success. In this study, we identified the C-terminal region (amino acids 3491 to 4701) of the V. vulnificus multifunctional autoprocessing RTX (MARTXVv or RtxA1) protein, RtxA1-C, as a promising antigen that induces protective immune responses against V. vulnificus. Vaccination of mice with recombinant RtxA1-C protein with adjuvant elicited a robust antibody response and a dramatic reduction in blood bacterial load in mice infected intraperitoneally. Vaccination resulted in significant protection against lethal challenge with V. vulnificus. Furthermore, intraperitoneal passive immunization with serum raised against the recombinant RtxA1-C protein demonstrated marked efficacy in both prophylaxis and therapy. These results suggest that active and passive immunization against the C-terminal region of the RtxA1 protein may be an effective approach in the prevention and therapy of V. vulnificus infections. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Vibrio vulnificus is a halophilic, gram-negative bacterium that is endemic to estuarine environments in temperate and tropical areas, and is a commensal bacterium in shellfish and fish [1–3]. Infection following ingestion of contaminated seafood or through a wound frequently results in septicemia in patients with hepatic diseases or immunocompromised conditions. Even with aggressive antibiotic treatment, fatality rates of over 50% have been observed within one or two days after the first symptoms appear [4–8]. Thus, given the rapid progression of disease and the ineffectiveness of existing therapies, there is a great need for effective measures for the prevention and control of V. vulnificus infection.

∗ Corresponding author at: Department of Microbiology and Immunology, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-756, Republic of Korea. Tel.: +82 63 270 3068; fax: +82 63 270 3066. E-mail address: [email protected] (K.M. Chung). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.11.019

Several virulence factors including capsular polysaccharide, iron assimilation systems, flagella, pili, VvhA, VvpE, and multifunctional autoprocessing repeats-in-toxin (RTX) (MARTXVv or RtxA1) have been identified and characterized as important factors in the pathogenesis of V. vulnificus [9,10]. These virulence factors play important pathological roles in bacterial attachment to host tissues, colonization, invasion, and survival [9,10]. Among those virulence factors, RtxA1, a secreted exotoxin, belongs to the RTX family produced by a broad range of pathogenic bacteria [9,11]. RtxA1 is a multifunctional toxin that is predicted to carry multiple domains associated with multiple cytotoxic and cytopathic activities including actin aggregation, necrosis, apoptosis, and induction of reactive oxygen species [12–16]. Additionally, recent findings suggest that the RtxA1 toxin is involved in protecting V. vulnificus from phagocytosis and is one of the major cytotoxins responsible for the high cytotoxicity of V. vulnificus [13,17]. In this regard, RtxA1 could be an attractive target for the development of specific preventive and therapeutic measures targeting the cytotoxicity of V. vulnificus. However, the usefulness of the RtxA1 protein as a potential vaccine antigen has not yet been reported.

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In this study, we demonstrated that the C-terminal region (amino acids 3491 to 4701) of the V. vulnificus RtxA1 protein, RtxA1-C, induced effective protective immunity against V. vulnificus infection by an intraperitoneal route. Passive immunization with anti-RtxA1-C serum prior to V. vulnificus infection also conferred protection. Furthermore, immune serum against RtxA1-C was effective in treating established V. vulnificus infection. Overall, these results demonstrate the potential for the C-terminal region of RtxA1 to serve as a highly protective antigen and a target for the development of an effective vaccine and therapeutic antibody against V. vulnificus. 2. Materials and methods 2.1. Recombinant RtxA1-C production in Escherichia coli A six-histidine-tagged C-terminal region (amino acids 3491 to 4701) of V. vulnificus RtxA1 protein, RtxA1-C, was expressed in E. coli and purified as previously described [18]. Briefly, the E. coli BL21(DE3)/pLys S strain (Invitrogen, Carlsbad, CA) transformed with pET-RtxA1 (3491–4701) plasmid was grown in LB, induced with 1.0 mM isopropyl thiogalactoside (IPTG), pelleted, and sonicated in 300 mM NaCl, 25 mM Tris-HCl (pH 7.5), 10% glycerol, and 0.1% Tween-20. After sonication, the six-histidine-tagged protein in soluble proteins was purified over a Ni-agarose affinity column according to the manufacturer’s instructions (QIAGEN, Palo Alto, CA) and by size-exclusion chromatography on a Hi-load 16/60 Superdex 200 column (Amersham Biosciences, Piscataway, NJ) in 150 mM NaCl and 25 mM HEPES (pH 7.4). The purified recombinant protein was concentrated and analyzed by SDS-PAGE followed by Coomassie blue staining. To generate six-histidine-tagged glutathione S-transferase (GST) as a control protein, the GST gene was amplified from the pGEX-KG plasmid (Novagen, Madison, WI) by PCR. The following oligonucleotides were used as forward- and reverse-primers: forward, 5 -GACGCTAGCGGATCCCCTATACTAGGTTAT-3 ; reverse, 5 -ACACTCGAGCTATCCATCCGATTTTGGAGGATGGTC-3 . The PCR fragment was digested and inserted into the NheI and XhoI sites of pRSET-A vector (Invitrogen) and sequenced. The six-histidinetagged GST protein was expressed, and then purified by the Ni-agarose affinity column and size-exclusion chromatography as described above. 2.2. Ethics statement for animal use Mouse experiments and protocols were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee at Chonbuk National University (Approved No. CBU 2013-0008). All mouse experiments were designed to minimize the number of animals used, and all efforts were made to minimize their suffering and distress. 2.3. Immunization and collection of antiserum All wild-type CD1 mice were purchased from a commercial source (Orient Bio Inc., a branch of Charles River Laboratories, Seongnam, Korea). For immunization, eight-week-old female CD1 mice were intraperitoneally primed and boosted at 3-week intervals with purified RtxA1-C (20 ␮g/mouse) or GST protein (20 ␮g/mouse) that was mixed with Monophosphoryl Lipid A (MPL) adjuvant (Sigma Adjuvant System; Sigma, St. Louis, MO). MPL has been administered as an adjuvant without a significant increase in reactogenicity in several clinical trials for vaccines against cancer, malaria, hepatitis B, and herpes simplex [19–24]. For the vaccination experiments in this study, the mice were boosted two times with RtxA1-C protein or GST control protein. For generation of

antiserum to study the prophylactic and therapeutic effects of antiRtxA1-C serum, the mice were boosted three times with RtxA1-C protein or GST control protein. To collect serum, blood was collected 14 days after the last boost by phlebotomy of the axillary vein prior to euthanasia. Following collection, blood was allowed to clot. After centrifugation at 4 ◦ C, serum was collected, aliquoted, and stored at −80 ◦ C. 2.4. Mouse experiments For bacterial infection, the V. vulnificus M06-24/O strain was cultured overnight in HI broth with 2.5% NaCl, and was then subcultured until the mid-log growth phase with agitation at 37 ◦ C. Next, the bacteria were washed three times with phosphate buffer saline (PBS) and were then resuspended to the appropriate concentration for infection. Eight-week-old CD1 mice were inoculated intraperitoneally with V. vulnificus. To evaluate the efficacy of RtxA1-C immunization against V. vulnificus infection, immunized mice were intraperitoneally inoculated with 8 × 106 colony-forming units (CFU) (∼14.5 LD50 ) of V. vulnificus at day 14 after the second immunization. For the anti-RtxA1-C serum transfer experiments, eight-weekold female CD1 mice were intraperitoneally infected with 2 × 106 CFU (∼3.6 LD50 ) of V. vulnificus at a given time point before or after administration with a single dose (200 ␮l) of antiserum harvested from RtxA1-C- or GST-immunized mice at day 14 after the third immunization. 2.5. Western blot analysis Equivalent amounts of protein were loaded and separated in 12% Tris-glycine polyacrylamide gel electrophoresis. After the proteins were transferred to nitrocellulose membranes (Amersham Biosciences), the membranes were treated with the blocking solution (5% (w/v) non-fat dry milk in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.025% Tween-20 (TBST)), washed in TBST, and incubated with a 1/2000 dilution of the anti-V. vulnificus serum from wild-type CD1 mice infected with 1 × 106 CFU (∼1.8 LD50 ) of V. vulnificus. After washing, membranes were subsequently incubated with a 1/3000 dilution of a secondary antibody conjugated with horseradish peroxidase (HRP), washed, developed with enhanced luminol-based chemiluminescent (ECL) Western blotting reagent (Amersham Biosciences), and detected with a luminescent image analyzer (LAS-1000; Fujifilm, Tokyo, Japan). 2.6. Enzyme linked immunosorbent assay Enzyme linked immunosorbent assay (ELISA) was used to determine the level of RtxA1-C-specific IgG, IgG1, IgG2a, or IgM in serum collected from V. vulnificus-infected mice and recombinant RtxA1-C-immunized mice [18,25]. Serum was collected from blood obtained by tail-vein nicking or phlebotomy of the axillary vein prior to euthanasia. In brief, microtiter plates (Maxi-Sorp; Nalge Nunc International, Rochester, NY) were coated overnight at 4 ◦ C with 55 ␮l per well of the purified recombinant RtxA1-C protein at 1 ␮g/ml. The coated plates were treated with blocking buffer (PBS, 0.025% Tween-20, 3% bovine serum albumin (BSA), and 0.025% NaN3 ), incubated with serial dilutions of serum, and washed with washing buffer (PBS, 0.025% Tween-20, and 0.5% BSA). Then, plates were incubated with biotin-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgM (Sigma) and HRP-conjugated streptavidin (Sigma) at 4 ◦ C. After extensive washing, enzyme reactivity was measured by adding 3,3 ,5,5 -tetramethyl-benzidine substrate, the reaction was terminated by adding an equal volume of 1 M H2 SO4 , and the optical

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density (OD) at 450 nm was determined. Naïve mouse serum was used as the negative control. The antibody titer in ELISA was calculated as the reciprocal of the last serum dilution equal to the mean OD of the negative control samples plus three times the standard deviation [25,26]. 2.7. Quantitation of blood bacterial load For determining the amount of live bacteria in the blood stream of RtxA1-C- and GST-immunized mice after infection, the immunized CD1 mice were infected with 8 × 106 CFU of V. vulnificus 14 days after the second immunization. We obtained whole blood samples by phlebotomy of the axillary vein at 150 min post-infection because high bacterial load in the blood was detected 90 min after the infection [13]. Counting of viable bacteria was performed using the plating method. 2.8. Statistical analysis All data were analyzed with Prism software (GraphPad Software Inc., San Diego, CA). For antibody titration and bacterial load experiments, statistical significance was determined using the Mann–Whitney test. For survival analysis, Kaplan–Meier survival curves were analyzed using the log-rank and Mantel–Haeszel tests. Relative percent survival was determined using the formula previously described by Amend [27]. 3. Results 3.1. Antibody response to RtxA1-C To evaluate whether an anti-RtxA1-C-specific antibody response occurred in mice infected with live V. vulnificus, mice were intraperitoneally infected with 1 × 106 CFU (∼1.8 LD50 ) of V. vulnificus. Three weeks post-infection, serum was obtained from surviving mice and ELISA was performed to determine specific immunoreactivity to RtxA1-C protein. Among the nine surviving mice of the 22 mice that were infected intraperitoneally, eight (88.9%) manifested high titers of anti-RtxA1-C-specific IgG ranging from 102 to 105 (Fig. 1). Also, six (66.7%) and five (55.6%) mice generated substantial increases (∼102 or higher) in anti-RtxA1-C IgG1 and IgG2a antibody titers, respectively (Fig. 1). Paired sera from non-infected mice did not recognize RtxA1-C protein in ELISA (data not shown). These results suggest that the antibody against RtxA1-C protein, the C-terminal region (amino acids 3491 to 4701) of the RtxA1 protein, could be induced by immune responses through both the Th1 and Th2 directions after an experimental infection with live V. vulnificus, indicating that the RtxA1-C protein has relevant antigenic properties as a potential vaccine candidate. Although the C-terminal region of RtxA1 of V. vulnificus has been identified as a possible antigen for the generation of monoclonal antibodies against RtxA1, there have been no reports evaluating the immunogenicity of the recombinant RtxA1 protein for vaccination [18]. To investigate the vaccine potential of the V. vulnificus RtxA1 protein, we expressed and purified the recombinant RtxA1-C protein which encompassed amino acids 3491 to 4701 of the 501 kDa RtxA1 toxin, and the glutathione S-transferase (GST) protein was used as a negative control (Fig. 2A). The purified RtxA1-C protein was confirmed by Western blotting with mouse serum against V. vulnificus (Fig. 2B). Then, wild-type CD1 mice were immunized and boosted at 3-week intervals with the purified recombinant RtxA1-C protein or GST control protein admixed with MPL adjuvant. On day 14 after the second immunization, sera were collected from the vaccinated mice and the titers of the RtxA1-C-specific

Fig. 1. Natural immune response against the recombinant RtxA1-C protein in live V. vulnificus-infected mice. CD1 mice were intraperitoneally infected with V. vulnificus. Titers of anti-RtxA1-C-specific IgG, IgG1, and IgG2a were determined by ELISA. ELISA was performed with purified recombinant RtxA1-C protein 21 days post-infection. The antibody titers are expressed by each filled square (IgG), circle (IgG1), and diamond (IgG2a) from an individual mouse. Solid dashes indicate the mean titers of antigen-specific IgG, IgG1, and IgG2a. The dotted line represents the limit of detection of the assay.

antibody were determined by ELISA. RtxA1-C-immunized mice showed anti-RtxA1-C IgG titers that were approximately 104 -fold higher compared to the GST-immunized mice (n = 6, P < 0.0001) (Fig. 2C). In addition, vaccination with the recombinant RtxA1-C protein induced significant increases in anti-RtxA1-C IgM titers compared with GST control-vaccinated mice (n = 6, P = 0.0001) (Fig. 2D). Overall, these results suggest that the recombinant RtxA1C protein effectively induced specific IgG and IgM humoral immune responses in mice. 3.2. Effect of recombinant RtxA1-C vaccination on survival and blood bacterial load We next evaluated whether RtxA1-C vaccination could provide protection against lethal V. vulnificus challenge. Eight-week-old CD1 mice were immunized with the RtxA1-C protein or GST control protein mixed with MPL adjuvant. Two weeks after the second boost, immunized mice were intraperitoneally challenged with 8 × 106 CFU (∼14.5 LD50 ) of live V. vulnificus. Notably, vaccination with the recombinant RtxA1-C antigen protected 93.3% (14 of 15) of mice from the lethal challenge (n = 15, P < 0.0001) and the relative percent survival was 92.9% (Fig. 3A). In contrast, the GST control protein did not confer significant benefit on survival, and only 6.7% of mice (1 of 15) survived. Since V. vulnificus infection causes septicemia [8], we evaluated the effect of RtxA1-C immunization on the blood bacterial load to better understand the mechanism of protection. Eight-week-old CD1 mice were immunized and challenged with live V. vulnificus as described above. Blood stream bacterial load was measured 150 min after the intraperitoneal infection. Interestingly, all (9 of 9) GST-vaccinated mice, but only 11.1% (1 of 9) of RtxA1-C-immunized mice had detectable levels of the bacteria in the blood, and the GST-treated mice had significantly higher bacterial counts (approximately 105 CFU/ml) than observed in RtxA1-C-immunized mice (n = 9, P < 0.0001) (Fig. 3B). Collectively, the protection and blood bacterial load analyses suggest that vaccination with the recombinant RtxA1-C protein is sufficient to provide protection against lethal V. vulnificus challenge by limiting bacterial load in the blood stream.

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Fig. 2. Purification and antibody responses elicited by the recombinant RtxA1-C protein. (A) Purification of recombinant RtxA1-C and GST control proteins. The E. coli-expressed RtxA1-C and GST proteins were purified after sequential Ni-agarose affinity and size-exclusion chromatography. The purified samples, GST (lane 1) and recombinant RtxA1-C (lane 2), were separated by 12% SDS-PAGE and stained with Coomassie blue. (B) Western blot analysis. GST (lane 1) and recombinant RtxA1-C (lane 2) were separated by 12% SDS-PAGE and were subjected to Western blot analysis using serum from a V. vulnificus-infected mouse. The recombinant GST and RtxA1-C protein are indicated by asterisk and solid arrowheads, respectively. The position of protein size markers (in kDa) is indicated on the left of the gel. (C-D) CD1 mice were immunized with the recombinant RtxA1-C protein or GST control protein plus adjuvant. Sera were obtained from the vaccinated mice on day 14 after the second immunization. Titers of IgG (C) and IgM (D) were determined by ELISA for binding to the purified recombinant RtxA1-C protein (55 ng per well). The data shown were from two independent experiments. Each open circle and filled square represents a sample from an individual mouse from GST control- and RtxA1-C-immunized mice, respectively. Solid lines indicate the mean titers of anti-RtxA1-C specific IgG and IgM. The dashed lines represent the limit of sensitivity of the assay.

3.3. Prophylactic and therapeutic effects of anti-RtxA1-C serum

4. Discussion

Although several previous studies have provided evidence that the RtxA1 exotoxin of V. vulnificus is a major virulence factor and is required for bacterial survival during infection [13–15,28], none of these studies have addressed whether passive transfer of immune serum against the RtxA1 protein could provide protection against V. vulnificus infection. To assess whether the anti-RtxA1-C antibody response was sufficient to confer protection and to explore whether anti-RtxA1-C antibody-based therapy could have an effect against V. vulnificus infections, we determined the efficacies of anti-RtxA1C serum in a mouse infection model. To evaluate the prophylactic efficacy of the anti-RtxA1-C serum, eight-week-old CD1 mice were challenged with 2 × 106 CFU (∼3.6 LD50 ) of V. vulnificus at 4.5 h after intraperitoneal administration of a single dose of anti-RtxA1-C or anti-GST serum (200 ␮l). A single dose of anti-RtxA1-C serum exerted 100% (10 of 10) protection against the lethal V. vulnificus infection, as compared to 10% (1 of 10) survival in the anti-GST serum-treated control group (n = 10, P < 0.0001) (Fig. 4A). Interestingly, anti-RtxA1-C-treated mice did not show any externally visible symptoms and completely recovered after 16 h of infection. Since the pooled anti-RtxA1-C serum showed outstanding prophylactic efficacy, we further evaluated its therapeutic activity in a pre-established V. vulnificus infection. Eight-week-old CD1 mice were intraperitoneally infected with 2 × 106 CFU (∼3.6 LD50 ) of V. vulnificus and then were treated with a single dose of anti-RtxA1-C or anti-GST serum (200 ␮l) 1 h after infection through the intraperitoneal route. A single dose of anti-RtxA1-C serum at 1 h after the bacterial challenge protected 100% (10 of 10) of mice (n = 10, P < 0.0001) (Fig. 4B). However, 40% (4 of 10) of those mice treated with the anti-RtxA1-C serum manifested symptoms of infection up until 16 h post challenge, which included fur ruffling and slightly reduced activity. These mice began to recover and were almost normal by 40 h post-infection. In contrast, only 10% (1 of 10) of the infected mice in the anti-GST control group survived, similar to what was observed in the prophylactic study. These results suggest that passive transfer of anti-RtxA1-C serum can provide sufficient prophylactic and therapeutic immunity against V. vulnificus.

V. vulnificus, a member of the normal marine microbiota, has emerged as one of the most virulent, life-threatening foodborne pathogens. Despite state-of-the-art therapeutic interventions, infection with V. vulnificus still leads to a high rate of mortality [5,9,29]. Therefore, finding an antigen that can induce a sufficiently protective immune response is critical. In this study, we evaluated whether the RtxA1 exotoxin, the most potent cytotoxin produced by pathogenic V. vulnificus, could serve as a vaccine antigen in a mouse model. We demonstrated that immunization with the C-terminal region (amino acids 3491–4701) of the RtxA1 protein, RtxA1-C, provided effective protection against V. vulnificus infection. Vaccination with the recombinant RtxA1-C protein in combination with MPL adjuvant produced a high titer antibody response and elicited protective immunity by significantly reducing bacterial load in the blood stream. A single-dose adoptive transfer of anti-RtxA1-C serum conferred successful prophylactic and therapeutic outcomes following V. vulnificus infection. There have been several attempts to develop vaccines against V. vulnificus, but no vaccine exists for human use. Formalininactivated whole cells and cell extracts of V. vulnificus have been tested as vaccine antigens, and showed substantial protective activity against the infection [30]. However, whole cells or bacterial cell extracts are seldom used in modern vaccines because of reactogenicity risks. V. vulnificus capsular polysaccharide (VvCPS) is a major virulence factor, and capsule loss by mutation results in avirulence [31–33]. VvCPS provides protective immunity (40–80% survival) when it is administered as a VvCPS-protein-conjugated vaccine [34]. Despite its dominant role in pathogenesis and its protective efficacy as a vaccine antigen, VvCPS is limited as a potential vaccine candidate; serotypes and carbotypes of VvCPS are extremely diverse, and cross-protective activity of anti-VvCPS serum is limited [32,35,36]. The VvpE protein, a metalloprotease secreted by V. vulnificus, was also reported to have elicited robust immune responses and conferred significant protection (75% survival) against the bacterial infection [37]. However, many doubts have been raised concerning the pathogenic role of VvpE. Deletion of the vvpE gene did not have any effect on the cytotoxicity and in vivo virulence of V. vulnificus [38–40]. Moreover, VvpE

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Fig. 3. Active immunization against V. vulnificus with the RtxA1-C vaccination. (A) Survival of RtxA1-C- and GST-vaccinated mice. Eight-week-old CD1 mice were vaccinated with recombinant RtxA1-C or GST control protein. The mice were intraperitoneally infected with 8 × 106 CFU (∼14.5 LD50 ) of V. vulnificus at day 14 after the second immunization. The survival curves were constructed using data from three independent experiments using five animals each. The survival rate for the RtxA1-C-immunized mice compared to that of the GST-immunized mice was significantly higher (n = 15, P < 0.0001). Open circles and filled squares indicate GSTand RtxA1-C-immunized mice, respectively. (B) Effect of RtxA1-C-vaccinated mice on blood bacterial load. Eight-week-old CD1 mice were vaccinated with recombinant RtxA1-C or GST control protein and were intraperitoneally inoculated with V. vulnificus as described above. Blood bacterial load was determined 150 minutes post-infection. The resulting bacterial load of RtxA1-C-immunized mice was significantly lower than for GST-immunized mice (n = 9, P < 0.0001). The data shown are the results from three independent experiments and three mice were used in each experiment. Each open circle and filled square indicates a sample from an individual mouse from GST- and RtxA1-C-vaccinated mice, respectively. Solid lines denote the average CFU per milliliter of blood in the GST- and RtxA1-C-vaccinated mice. The dashed line represents the limit of sensitivity of the assay.

raises safety concerns over the possibility of incomplete inactivation and toxicity since the injection of purified VvpE leads to tissue necrosis and cutaneous lesions [41–43]. The use of RtxA1 exotoxin could avoid these potential concerns because RtxA1 is significantly induced after contact between V. vulnificus and host cells, and causes necrotic cell death in a contact-dependent manner [13]. In addition, the recombinant RtxA1-C protein did not show any cytotoxicity to in vitro cultured eukaryotic cells (data not shown) and is strictly conserved in clinical isolates [44,45]. Given these findings, vaccination with the C-terminal fragment (amino acids 3491–4701) of the RtxA1 protein, RtxA1-C, is likely to be safe and practical. Furthermore, high titers of anti-RtxA1-C IgG antibody were detected in most V. vulnificus-infected mice (Fig. 1), suggesting that RtxA1 is a target of adaptive humoral immunity after natural infection. Similar to all MARTX toxins, V. vulnificus RtxA1/MARTXVv harbors conserved repeat regions at the N and C termini and multiple activity domains in the central portion that include a Rhoinactivation domain (RID), an actin cross-linking domain (ACD),

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Fig. 4. Prophylactic and therapeutic efficacy of anti-RtxA1-C serum in a lethal V. vulnificus challenge model. (A) Prophylactic efficacy of anti-RtxA1-C serum. Eightweek-old CD1 mice were intraperitoneally administered a single dose of sera obtained from RtxA1-C- or GST control protein-immunized mice. Then, the mice were intraperitoneally infected with 2 × 106 CFU (∼ 3.6 LD50 ) of V. vulnificus 4.5 h later. Anti-RtxA1-C serum provided significant protection compared to treatment with anti-GST control serum (n = 10, P < 0.0001). (B) Therapeutic efficacy of antiRtxA1-C serum. Eight-week-old CD1 mice were intraperitoneally inoculated with 2 × 106 CFU (∼ 3.6 LD50 ) of V. vulnificus and a single dose of anti-RtxA1-C or anti-GST serum was administered intraperitoneally 1 h after bacterial infection. The single dose of anti-RtxA1-C serum after infection resulted in effective post-exposure therapy compared to administration of anti-GST control serum (n = 10, P < 0.0001). All survival curves were constructed using data from two independent experiments with five animals each. Open circles and filled squares indicate anti-GST and antiRtxA1-C-treated mice, respectively.

and a cysteine protease domain (CPD) [11,46]. Upon encountering a eukaryotic cell, the N and C termini of the toxin insert into the plasma membrane and likely form a pore to transfer the toxin’s central portion into the cytosol [47]. After translocation, the central region of the large toxin is cleaved into effector domains that access intracellular targets [48]. However, the detailed functions of the extracellular RtxA1 domain have not been well characterized. Based on the reduction of blood bacterial load in the RtxA1-C vaccinated mouse (Fig. 3B), we speculated that the RtxA1-C region with the CPD and the glycine-rich repeat region in the C-terminus of the secreted RtxA1 might be associated with dissemination of the infecting bacteria into the bloodstream. Although more studies are required to characterize, in detail, how and at what stage the RtxA1C vaccine acts, we hypothesize that the antibody response induced following vaccination might limit V. vulnificus invasion and/or trigger clearance of V. vulnificus from the bloodstream. This could occur by inhibiting colonization and enhancing phagocytosis of the bacteria, which might result in decreased spread to the liver and other organs, and would enhance survival by blocking fulminant systemic infection. In support of this, the RtxA1/MARTXVv -deficient mutants of V. vulnificus were more sensitive to phagocytosis and

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