Epidemiological survey of serum titers from adults against various Gram-negative bacterial V-antigens.

The V-antigen, a virulence-associated protein, was first identified in Yersinia pestis more than half a century ago. Since then, other V-antigen homologs and orthologs have been discovered and are now considered as critical molecules for the toxic effects mediated by the type III secretion system during infections caused by various pathogenic Gram-negative bacteria. After purifying recombinant V-antigen proteins, including PcrV from Pseudomonas aeruginosa, LcrV from Yersinia, LssV from Photorhabdus luminescens, AcrV from Aeromonas salmonicida, and VcrV from Vibrio parahaemolyticus, we developed an enzyme-linked immunoabsorbent assay to measure titers against each V-antigen in sera collected from 186 adult volunteers. Different titer-specific correlation levels were determined for the five V-antigens. The anti-LcrV and anti-AcrV titers shared the highest correlation with each other with a correlation coefficient of 0.84. The next highest correlation coefficient was between anti-AcrV and anti-VcrV titers at 0.79, while the lowest correlation was found between anti-LcrV and anti-VcrV titers, which were still higher than 0.7. Sera from mice immunized with one of the five recombinant V-antigens displayed cross-antigenicity with some of the other four V-antigens, supporting the results from the human sera. Thus, the serum anti-V-antigen titer measurement system may be used for epidemiological investigations of various pathogenic Gram-negative bacteria.

Introduction

The type III secretion system (TTSS) plays a major role in the virulence of many Gram-negative bacteria [1-3]. Through the TTSS, Gram-negative bacteria inject their effector molecules to target eukaryotic cells and induce a favorable environment for their infections. Translocation is a mechanism through which effector molecules of the TTSS pass through the targeted eukaryotic cell membrane. During translocation, three bacterial proteins form a translocational pore structure called the ‘translocon’ [1-3]. A cap protein in the secretion apparatus of the type III secretion needle is one type of translocon protein, which is called the V-antigen in Yersinia spp. for historical reasons [4-8]. Briefly, the V-antigen, a virulence-associated protein, was identified as an antigenic component recognized by the immune system in Yersinia pestis plague-infected hosts more than half a century ago [4-8]. In the 1980s, the V-antigen of Y. pestis was identified as a low calcium response (lcr)-associated protein (named LcrV) encoded in the plasmid associated with its virulence [9]. A homologous gene called PcrV was identified in the Pseudomonas aeruginosa genome [10]. It has been reported that the virulence-associated with the TTSS can be inhibited by a specific antibody against LcrV in Yersinia and PcrV in P. aeruginosa [11, 12]. Because vaccinating mice with LcrV or PcrV has protective effects against lethal infections by Yersinia or P. aeruginosa, respectively, anti-PcrV immunotherapies were developed to target human infections with P. aeruginosa using immunoglobulins [13-24] and vaccines [25-27], from which several projects have progressed to human clinical trials [28-31].

We recently published an epidemiological study on serum titers against PcrV in human volunteers [32], and another showing how prophylactic administration of human serum-derived immunoglobulin with a high anti-PcrV titer significantly improves the survival rate, pulmonary edema, and inflammatory cytokine production of a P. aeruginosa pneumonia model [18]. The results of both studies imply that immunity against the V-antigen and its homologs might be necessary to prevent infections caused by pathogenic bacterial species employing the TTSS-virulence mechanism [18, 32]. V-antigen homologs have been recently reported in several Gram-negative bacteria, including Aeromonas spp., Vibrio spp., and Photorhabdus luminescens (hereafter referred to as Ph. luminescens) [33]. Although specific immunity against the V-antigen or its homologs appears to be important for host immunity against such bacterial infections, insufficient information is available on human immunity against V-antigens. Therefore, here, we conducted an epidemiological study on serum titers against the V antigen and its homologs in Y. pestis, Aeromonas salmonicida, Vibrio parahaemolyticus, and Ph. luminescens. Potential associations in terms of age, titer levels, and cross-reactivity were evaluated among the recombinant V-antigen homologs. Moreover, for some species, the titer levels against these antigens were highly correlated, and some V-antigen homologs showed cross-reactivity.

Materials and methods

Construction of five recombinant Gram-negative bacterial V-antigens (PcrV, LcrV, AcrV, VcrV, and LssV) and P. aeruginosa porin F from the outer membrane (OprF)

Five recombinant V-antigens and recombinant P. aeruginosa OprF were constructed. Details on the PCR primers and cloning sites are listed in . The coding regions of the V-antigens were amplified by polymerase chain reaction (PCR) with specific primers containing restriction enzyme sites for insertion into a protein expression vector. PCR-amplified genes were cloned into the pCR2.1 cloning vector and E. coli TOP10F cells via TOPO cloning (Thermo Fisher Scientific, Waltham, MA, USA). After digesting the purified plasmids containing each individual cloned gene with restriction enzymes, the inserted coding regions of each gene were transferred to the multiple cloning site of the expression vector pQE30 (Qiagen, Hilden, Germany) for expression of a hexahistidine-tagged protein in E. coli M15. The various endotoxin-free Gram-negative bacteria V-antigens were prepared as reported previously () [17, 20].

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of extracted recombinant hexahistidine-tagged V-antigen proteins.

Recombinant PcrV from P. aeruginosa, LcrV from Y. pestis, AcrV from Aeromonas salmonicida, VcrV from V. parahaemolyticus, and LssV from Ph. luminescens were separated by SDS-PAGE using a 10% Bis-Tris-gel.

Survey participants and study background

This study was approved by the Ethics Committee of Kyoto Prefectural University of Medicine (Approval number: RBMR-E-326-1; Kyoto, Japan). As a non-interventional and non-invasive retrospective observational study, the need for consent was waived by the ethics committee. Adult patients (n = 186) who underwent anesthesia in the Central Operating Division of Kyoto Prefectural University of Medicine, from April 2012 to March 2013, participated in this study as volunteers, as reported previously [32]. Briefly, serum was prepared from the remaining small amount of each blood sample collected for arterial blood gas analysis for induction of anesthesia and then stored at −80°C.

Anti-V antigen titer measurements

We developed an enzyme-linked immunoabsorbent assay (ELISA) to measure serum anti-V antigen titers. Microwell plates (Nunc C96 Maxisorp; Thermo Fisher Scientific) were coated for 2 h at 4°C with recombinant V-antigen proteins (recombinant PcrV from P. aeruginosa, LcrV from Yersinia, AcrV from Aeromonas, VcrV from Vibrio, and LssV from Ph. luminescens) suspended in coating buffer (1 μg/mL in coating solution; 0.05 M NaHCO3, pH 9.6) [32] (). The plates were washed twice with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (P9416; Sigma-Aldrich, St. Louis, MO, USA) and then blocked with 200 μL of 1% bovine serum albumin/PBS overnight at 4°C. Samples (serial dilution: 1280×) were applied to the plates (100 μL/well) and then incubated overnight at 4°C. Peroxidase-labeled anti-human IgG (A8667, Sigma- Aldrich) was applied at 1:60,000 for 1 h at 37°C. After six washes, the plates were incubated with 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (A3219; Sigma-Aldrich) at room temperature for 30 min. After adding 0.5 M H2SO4 at 100 μL/well to the plates, the optical density (OD) at 450 nm was measured with a microplate reader (MTP-880Lab; Corona Electric, Hitachinaka, Japan). To ensure no cross-reactivity with Escherichia coli proteins, we performed an inhibition ELISA with a soluble fraction of E. coli M15 lysate, and no significant effect on titer measurement was observed. Except for human monoclonal anti-PcrV IgG mAb 6F5 as a standard to measure the anti-PcrV titer [32], there is no human anti-V-antigen IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the mAb 6F5 standard [32], the OD measured under a consistent condition with the same secondary antibody was used to evaluate the titers.

Inhibition ELISA

Cross-reactivity was analyzed by an inhibition ELISA. Two human sera with relatively high anti-PcrV, LcrV, AcrV, VcrV, and LssV titers were diluted at 1:1000 and preincubated with either recombinant LssV or recombinant OprF (100 μg/mL) overnight at 4°C. The anti-V-antigen titers were measured in triplicate by an ELISA using recombinant V-antigen-coated plates.

Immunizing mice with the V-antigens

Certified pathogen-free, male ICR mice (4 weeks old) were purchased from Shimizu Laboratory Supplies, Co, Ltd (Kyoto, Japan). Mice were housed in cages with filter tops under pathogen-free conditions. The protocols for all animal experiments were approved by the Animal Research Committee of Kyoto Prefectural University of Medicine before undertaking the experiments (Authorization number: M29-592). Three mice per group were intradermally immunized with one of the five recombinant V-antigen proteins (10 μg/dose) adjuvanted with complete Freund’s adjuvant in the first injection, and four weeks later with incomplete Freund’s adjuvant for the second injection. Eight weeks after the first injection, the immunized mice were euthanized with a large dose intraperitoneal injection of sodium pentobarbital, and peripheral blood samples were collected. Serum titers against the five V-antigens were individually measured by ELISAs, as described above.

Phylogenetic and cluster analyses

The five V-antigens were phylogenetically analyzed using ClustalW (Genome Net, https://www.genome.jp/tools-bin/clustalw) or RStudio (version 1.2, RStudio, Boston, MA, USA. https://www.rstudio.com) with R version 3.6.1 (The R Foundation, https://www.r-project.org). Unrooted trees were prepared using the neighbor-joining method, and rooted trees were prepared using the unweighted pair group method with arithmetic means applied to the ClustalW site and the standard R function plot.hclust. Heat maps where the individual values contained in a matrix are represented as colors with dendrograms prepared using the function phylo of the package “ape” for phylogenies (http://ape-package.ird.fr.) and the function heatmap.2 of the package gplots. The predicted three-dimensional structures were generated with the Cn3D macromolecular structure viewer at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml).

Statistical analysis

We performed a regression analysis of the measured antibody titers using the regression data analysis tool of Microsoft Excel for Mac (ver. 16.16.5, Microsoft Co., Redmond, WA, USA). Statistical analyses were conducted using InStat version 4.0 (GraphPad Software Inc., La Jolla, CA, USA). P-values were calculated using the Mann-Whitney U-test. A p-value of less than 0.05 was considered statistically significant.

Results

Anti-V antigen titers and volunteer age distribution

The 186 volunteers included 111 (59.7%) males and 75 (40.3%) females and an age distribution of 20–29 years (13, 7.0%), 30–39 years (20, 10.8%), 40–49 years (26, 14.0%), 50–59 years (23, 12.4%), 60–69 years (40, 21.5%), 70–79 years (42, 22.5%), and ≥80 years (22, 11.8%). No study participant had an active infection. The titers against the V-antigen across the age distribution of the 186 participants are shown in . There was no statistically significant correlation in the linear regression between age and each anti-V antigen titer, although high anti-PcrV titers were more common at over 50 years of age in the population, as reported previously [32]. As an overall trend, two separate titer peaks in the 40–49 and 70–79 age groups were observed in the age distribution of all anti-V-antigen titers.

Age distribution of human V-antigen titers.

The diluted serum (1,280×) was applied to ELISAs, and OD 450 nm values were measured. OD values of ≥0.5 are indicated by red dots, while yellow dots represent values between 0 and 0.3. OD: Optical density.

Correlations among the anti-V antigen titers

We next analyzed whether any correlations existed among the five anti-V antigen titers. The anti-LcrV and the anti-AcrV titers showed the highest correlation with a correlation coefficient of 0.84, followed by the anti-AcrV and anti-VcrV titers at 0.79, and the anti-LcrV titers and anti-VcrV titers at 0.74 () Low correlations were detected between anti-PcrV and any of the other anti-V-antigens.

Next, the inhibition ELISA was performed to show cross-antigenicity among V-antigens. As an irrelevant non-crossreactive protein, we prepared P. aeruginosa recombinant OprF using the same E.coli-derived recombinant protein construction system. The sequence alignment scores obtained from ClustalW between OprF and V-antigens were 9.8–11.4, whereas those among V-antigens were from 21.3 (between VcrV and AcrV) to 48.3 (between LcrV and LssV). Two human sera diluted 1000× were preincubated with either recombinant LssV or recombinant OprF overnight. Then, preprocessed serum titers against each V-antigen were measured in comparison with the titer levels of the original sera (). As a result, preincubation with OrpF did not affect the specific titer levels. However, preincubation with LssV decreased the titer levels compared with the titer levels of the original sera (*p<0.05 for AcrV, VcrV, and LssV).

Cluster analysis of the correlation coefficient values was conducted, and phylogenetic trees and a heat map were constructed (). The unrooted and rooted phylogenic trees and heat map showed that anti-PcrV had a unique profile among the five anti-V-antigen titers. The anti-LssV titer was located between anti-PcrV and the three other anti-V-antigen titers. Higher homology in terms of antigenicity was observed among anti-AcrV, anti-LcrV, and anti-VcrV titers.

Phylogenetic trees and heat maps showing correlations among the five anti-V-antigen titers.

A. Human serum titers. The correlation coefficients of the serum titer correlations shown in were matrixed. Phylogenetic trees (an unrooted tree, neighbor-joining method, and a rooted tree, unweighted pair group method with arithmetic mean) and a heat map demonstrating correlation coefficient values of the serum titer plots (Fig 3) as a color-index were constructed. B. Anti-V-antigen serum titers from mice immunized with one of the five V-antigens. The mean values from triplicate measurements were applied to construct a phylogenetic tree (unrooted tree, neighbor-joining method, and a rooted tree, unweighted pair group method with the arithmetic mean) and a heat map demonstrating O.D 450 nm values of the serum titers to the V-antigens as a color-index. OD: Optical density.

Fig 3

Serum titer correlations between two V-antigens.

The diluted serum (1,280×) was applied to ELISAs, and OD values at 450 nm were measured. The titer correlation of the two V-antigens was mapped in an X–Y plot. OD: Optical density.

Inhibition ELISA.

Anti-V-antigen titers of human sera (n = 2), which showed relatively high titers against all five V-antigens, preincubated with either recombinant LssV or OprF were measured in comparison with the original titer levels of the sera. *p < 0.05 with the Mann-Whitney U-test between mean values of original serum titers and those of titers after pre-inhibition with recombinant proteins. OD: Optical density.

Cross-reactive antibodies against V-antigens and anti-V antigen titers in serum from immunized mice

To evaluate potential cross-antigenicity among the five V-antigens, the serum titers from a mouse immunized with one of the five recombinant V-antigen proteins were measured by ELISAs. Cluster analysis of the OD values from the ELISA was performed, from which phylogenetic trees and a heat map were constructed (). LssV and AcrV showed higher cross-antigenicity with each other, unlike VcrV that was less cross-reactive with the other V-antigens (). Whereas the sera from the AcrV-immunized mice reacted with PcrV, the sera from PcrV-immunized mice did not react with AcrV.

Next, to determine whether correlations among the V-antigens had some association with the identity of the primary protein sequences, we investigated the identity of these sequences in the five V-antigens using the BLOSUM substitution score matrix in ClustalW (). In this map, the overall similarities of two out of five of the V-antigens for the whole molecules ranged between 21% and 49%. The similarity of the amino-terminal domain (14%–51%) and central domains (14%–45%) was low in comparison with the carboxyl-terminal domain that showed 48%–84% similarity. VcrV was unique with a long additional sequence (>160 amino acids, aa) at the amino-terminal domain and an additional sequence of 80 aa in the central domain of the sequence alignment.

ClustalW sequence alignment of the five primary V-antigen sequences.

The overall sequence similarities of two of the five V-antigens across the whole molecules were between 21% and 49%. The similarity of the amino-terminal domain (14%–51%) and central domains (14%–45%) was low in comparison with the carboxyl-terminal domain that had a 48%–84% similarity range. VcrV was unique with a long additional amino-terminal domain sequence (>160 aa) and an additional sequence (80 aa) in the central domain of the sequence alignment.

We also constructed phylogenetic trees of the V-antigens based on the primary amino acid sequence similarity scores of whole molecules, amino-terminal domains, central domains, and carboxyl-terminal domains (). In the cluster analysis of whole molecules, the similarity score was the highest between LcrV and LssV (48.6%) (). The amino-terminal domains showed similarity scores of <30%, except for the 51.9% similarity between LcrV and LssV (). For the center domains, the highest similarity score was obtained between AcrV and LssV (44.5%) (). In the cluster analysis of the carboxyl-terminal domains with the highest similarity scores (48%–84%) for their amino acid sequences, AcrV, LssV, and PcrV were closer than VcrV and LcrV ().

Phylogenetic trees and heat maps based on the primary amino acid sequences (whole molecule, amino-terminal, center, and carboxyl-terminal) of the V-antigens.

Phylogenetic analyses of the primary amino acid sequences of whole molecules, amino-terminal, center, and carboxyl-terminal domains were performed. The sequence alignment scores obtained from ClustalW are shown. Phylogenetic trees (an unrooted tree, neighbor-joining method) and a heat map demonstrating sequence alignment scores between two V-antigens as a color-index were constructed. A. Complete primary sequence. B. Amino-terminal domain, C. Center domain, D. Carboxyl-terminal domain. The amino acid positions in the amino-terminal, center, and carboxyl-terminal domains are as follows: LcrV: #1–#164, #165–#278, and #279–#326 respectively; PcrV: #1–#142, #143–#256, and #257–#294, respectively; AcrV: #1–#162, #163–#316, and #317–#361, respectively; VcrV: #1–#361, #362–#558, and #559–#607, respectively; LssV: #1–#170, #171–#280, and #281–#325, respectively.

These phylogenetic analyses using the primary amino acid sequences did not match well with the phylogenetic trees constructed from the correlations among the serum IgG titers of the five V-antigens (). In particular, VcrV, which has an extremely long central domain containing regions that are missing in the other V-antigens, occupied a separate position in the phylogenetic trees constructed from the primary amino acid sequences. Therefore, these findings suggest the titer cross-antigenicity in human sera may not be correlated with the similarity in primary amino acid sequences.

As we have reported previously, a blocking monoclonal antibody called Mab166 against PcrV recognizes the conformational structure, but not the primary amino acid sequence [15]. As shown in , the predicted three-dimensional structures of the V-antigens had similar dumbbell-like structures with two globular domains on either end of a grip formed by two coiled-coil motifs [37]. The grip that connected the two globular domains contained an antiparallel coiled-coil structure comprising central and carboxyl-terminal coiled-coil regions. The carboxyl-terminal was folded into a single long α-helix. Regarding the blocking antibody recognizing the three-dimensional conformational epitopes and not the primary amino acid sequences, not only the serum titer levels, but also the specific components that bind to the specific blocking regions in V-antigen molecules may be important to prevent the pathogenesis associated with TTSS virulence.

Predicted three-dimensional V-antigen structures.

The predicted three-dimensional structures were generated by the Cn3D macromolecular structure viewer at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml).

Discussion

Yersinia LcrV has been recognized as a V-antigen with immunoprotective characteristics in Yersinia infections since the 1950s [4, 5, 7, 8]. However, after almost 50 years, LcrV, P. aeruginosa PcrV, and Aeromonas AcrV were anatomically visualized as distinct structures on the tip of the needle of the injectisome of the type III secretion apparatus [38-41]. Because specific antibodies binding to a particular portion of this structure inhibit the translocation of type III secretory toxins in Yersinia and P. aeruginosa [11, 42], gaining a better understanding of the interactions between V-antigens and the host humoral immunity against the virulence of bacterial type III secretion is important to develop potential non-antibiotic treatments for infections in various hosts.

Cross-antigenicity among Yersinia spp. was reported nearly 40 years ago. In 1980, cross-immunity to Y. pestis was noted in mice that had been orally infected with Y. enterocolitica serotype O3 [43]. In 1983, it was also reported that partial protection in mice against Y. pestis infection by the Y. enterocolitica V-antigen was linked to the partial cross-reactivity of V-antigens [44]. Later, DNA sequencing of the most common serotypes of human pathogenic Y. enterocolitica and Y. pseudotuberculosis revealed two evolutionary distinct types of V-antigen in Yersinia spp. [45]. One type is represented by Y. enterocolitica serotype O8 strains WA, WA-314, and NCTC 10938 (LcrV-YenO8), whereas the other type is represented by Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O3, O9, and O5 (LcrV-Yps) [45]. By raising monospecific antisera against both types of V-antigen (Y. enterocolitica serotypes O3 and O8), anti-V-antigen serum was protective only when the immunizing V-antigen was the same type as the V-antigen produced by the infecting strain. The difference in protective immunity between the two types was caused by the presence of a hypervariable region between amino acids 225 and 232 [45]. The protectivity of the V-antigen was later confirmed and refined using the recombinant V-antigen of Y. pseudotuberculosis and monospecific anti-V-antigen serum [46]. In this previous study, antiserum against the Y. pseudotuberculosis V-antigen provided mice with passive immunity to challenge infection with Y. pestis or Y. pseudotuberculosis, but not Y. enterocolitica O8 (strain WA). These previous studies on cross-antigenicity to Yersinia V-antigens in mice imply that the structure of the critical domain, but not the overall primary amino acid sequence similarity per se, is important for protective immunity.

Other than Yersinia LcrV, only two studies of P. aeruginosa PcrV have reported antibody titers against P. aeruginosa PcrV in human serum [32, 47]. No other studies investigating antibody titers against other V-antigens have been reported to date. V. parahaemolyticus, a Gram-negative marine bacterium, of which the V-antigen titers were examined in the present study, causes food-borne gastroenteritis [48, 49]. Among Vibrio spp., V. harveyi (a Gram-negative bioluminescent marine bacterium) is ubiquitous in the marine environment. It is considered as one of the important bacterial species that form the normal flora of healthy shrimp, and its genome carries a V-antigen homolog gene [33, 50]. This bacterium is sometimes recognized as causing high mortality of shrimps in the worldwide shrimp fishing industry [51, 52]. As a halophilic Vibrio species, V. alginolyticus, which causes wound infections, was first recognized as pathogenic to humans in 1973 [53]. Recent studies have proposed that V. alginolyticus possesses the same TTSS gene organization as V. parahaemolyticus and V. harveyi [54, 55]. Aeromonas spp., such as A. salmonicida and A. hydrophila, are not pathogenic to humans, but cause infections in salmon and trout, and carry the AcrV V-antigen homolog [33]. However, A. hydrophila sometimes causes gastroenteritis in humans who acquire such infections by ingesting food or water containing sufficient numbers of this organism [56-58]. Ph. luminescens (a gammaproteobacterium in the Enterobacteriaceae family) is also not pathogenic to humans, but is a lethal pathogen of insects and possesses a pathogenicity island encoding the TTSS including the LssV V-antigen homolog [59-62]. It lives in the gut of an entomopathogenic nematode in the Heterorhabditidae family [33]. However, human infections with Photorhabdus spp. have recently been reported in the USA and Australia, suggesting that these bacteria are emerging human pathogens [63]. In comparison with the infections caused by P. aeruginosa, infections caused by Y. pestis or V. parahaemolyticus are less common. Therefore, it is interesting that titers against V-antigens from non-human pathogens were also detected in this study. Anti-AcrV showed a high correlation with anti-LcrV, and some correlation was detected between anti-LssV and anti-VcrV. The titers against the antigens from non-human pathogens might result from cross-antigenicity among the V-antigen homologs. Our cluster analysis with the heat map of anti-V-antigen titers from the 186 adult volunteers displayed a higher correlation between LcrV, AcrV, and VcrV than the correlation between PcrV and LssV and the other V-antigens ().

Immunizing mice with one of the five recombinant V-antigens resulted in cross-antigenicity of the sera against the V-antigens (Figs and . This result shows that VcrV is unique, while AcrV, LssV, and LcrV share some degree of cross-antigenicity with each other, although the results differed slightly from the cross-antigenicity observed with human sera. In our previous study on anti-PcrV titers in human sera, administration of extracted IgG derived from high titer anti-PcrV sera protected against challenge with lethal pneumonia in a murine model [18]. In this study, among the 198 volunteer-derived sera [32], the top 10 high titer sera against PcrV (greater than or equivalent to 0.5 at OD 450 nm) were used. Although we have human monoclonal IgG specific against PcrV as a standard for anti-PcrV measurement, no such human standard IgG against the other four V-antigens are available to date. It is difficult at this time to clearly evaluate where the level of protection is sufficient, but as many as 5% of the volunteers showed protective levels in our previous study [18], and the cross-antigenicity among the five V-antigens probably has a certain level of clinical significance in human immunity. Therefore, despite no similar immunological experiments being performed with other V-antigens, our study has shown that the cross-reactive antigenicity we observed among the various V-antigens with serum-specific anti-V antigen titers may afford some degree of immunological protection against various Gram-negative bacteria. Further investigation of the conformational blocking epitopes in the needle cap structure of type III secretory apparatus and the immunological aspects of the structural antigenicity of a critical portion of the V-antigens should provide a better understanding of how to effectively block the TTSS-associated virulence associated with various Gram-negative pathogens.

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12 Nov 2019

PONE-D-19-20754

Epidemiological survey of serum titers from adults against various Gram-negative bacterial V-antigens

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I have read the journal's policy and the authors of this manuscript have the following competing interests: T. Sawa has patents associated with PcrV immunization (World Patent No. WO0033872; European Patent No. EP1049488; U.S. Patent No. 6309651; U.S. Patent No. 6827935, and Japan Patent No. 2017-020501). Until 2011, T. Sawa received a patent fee from the Regents of the University of California related to the development of a therapeutic monoclonal antibody at KaloBios Pharmaceutical. There are no current financial relationships existing with any organization associated with this study.

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

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Reviewers' comments:

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, Kinoshita et al tried to establish serum anti-V antibody titer as method for the epidemiological investigations on various pathogenic Gram-negative bacteria. Authors developed an enzyme-linked immuno-sorbent assay to measure titers against each V antigen in the sera collected from 186 adult volunteers. However, the manuscript is identified with a rather confusing experimental approaches for epidemiological investigation of Gram-negative bacterial pathogens. The application of purified V antigens (PcrV from Pseudomonas aeruginosa, LcrV from Yersinia, LssV from Photorhabdus luminescens, AcrV from Aeromonas salmonicida and VcrV from Vibrio parahaemolyticus) and their cross reactivity in both human and mouse serum samples demonstrating the inability of this system to determine accurately the bacterium elicited antigenicity in human samples. The manuscript is also suffering with the complete lack of standard or control samples in entire experiments and that raised serious question against reproducibility of work.

Authors must include the closely related species and or strains of all five bacteria used in this study to validate it further.

Reviewer #2: Major comments:

1) Figure 4A doesn't match to the written part that describe it and actually it is identical to figure 7A. Please correct the figure.

2) There is no statistical method section in the Materials and Methods part so it is impossible to determine statistical significance, power analysis etc. Please include such a section with the relevant statistical analyses.

3) Regarding the heat maps, please provide a valid statistical/numerical information that defines the various colors. I'm sure that this data is provided by the relevant analysis tools. Please include a short description also in the figure legend.

Minor comments:

1)The sentence in line 273 is not clear.

2)Line 30; a dot is missing after the 0.7

3)Line 117; please correct the 0.0M to a valid value.

4)Line 119; please correct the 200mL to 200μL

5)Line 121; please correct the 100mL/well to 100μL/well

6)line 125; please correct the 100mL/well to 100μL/well

7)line 224; please correct "..carboxyl domains.." to carboxyl-terminal domains

Reviewer #3: This paper by Kinoshita et. al. looks into the antibody response to various bacterial V-antigens in a cohort of healthy adults. They find that many people have various antibody responses to the V-antigens and that many of these antibodies may cross-react. The data is sound and the authors are careful not to overstate their findings. I do have a few comments however;

1. It is not made clear what percent of people have reactive antibodies to each protein. This could be a percent of patients over a specified cutoff. You already have both ‘red’ and ‘orange’ cutoffs so these could be used.

2. Both the correlations in Figure 3 and the mice vaccination studies strongly suggest the presence of some cross-reactive antibodies in the human sera. This would be fairly easy to prove in the human sera by absorption experiments. Take a human sera with responses to multiple proteins- adsorb against the highest reacting protein and determine if this also removes response to the other V-antigens.

3. Figure 5 would be easier to interpret if it was a graph or heat map of the response levels instead of a photo of the plate. I can’t tell by eye if one well is more blue than another.

4. Lines 244-248 the authors state that the amino-acid sequences phylogeny in Fig 7 do not match the serum trees in Fig 4A. To my eye they do match pretty well especially 7A and 7B. In both LssV is closest to LcrV and VcrV is furthest away. Can the authors clarify their statement?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: 11-3-19 plos one review.docx

Click here for additional data file.

22 Dec 2019

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[Response]

I followed the above guidance.

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[Response]

We provide an original uncropped and unadjusted SDS-PAGE image of Fig.1, and wrote it in our cover letter for this resubmission.

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[Response]

We added the following:

Page 8, line 104 (page 8, line 139 of tracked_change_version): “As a non-interventional and non-invasive retrospective observational study, the need for consent was waived by the ethics committee.”

4. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript.

[Response]

We added the following:

Page 11, line 152 (page 11, line 214 of tracked_change_version):” …., the immunized mice were euthanized with a high dose intraperitoneal injection of sodium pentobarbital,”

5. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:

https://doi.org/10.1111/1348-0421.12353

https://doi.org/10.1111/1348-0421.12147

https://iai.asm.org/content/iai/65/2/446.full.pdf

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed.

[Response]

We provided the correct reference numbers in all the sentences which we cited.

6. Thank you for stating the following in the Competing Interests section:

I have read the journal's policy and the authors of this manuscript have the following competing interests: T. Sawa has patents associated with PcrV immunization (World Patent No. WO0033872; European Patent No. EP1049488; U.S. Patent No. 6309651; U.S. Patent No. 6827935, and Japan Patent No. 2017-020501). Until 2011, T. Sawa received a patent fee from the Regents of the University of California related to the development of a therapeutic monoclonal antibody at KaloBios Pharmaceutical. There are no current financial relationships existing with any organization associated with this study.

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Response]

We added the sentence as follows, in the section Conflict of Interest:

This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Responses to Reviewer comments:

Reviewer #1: In this manuscript, Kinoshita et al tried to establish serum anti-V antibody titer as method for the epidemiological investigations on various pathogenic Gram-negative bacteria. Authors developed an enzyme-linked immuno-sorbent assay to measure titers against each V antigen in the sera collected from 186 adult volunteers. However, the manuscript is identified with a rather confusing experimental approaches for epidemiological investigation of Gram-negative bacterial pathogens. The application of purified V antigens (PcrV from Pseudomonas aeruginosa, LcrV from Yersinia, LssV from Photorhabdus luminescens, AcrV from Aeromonas salmonicida and VcrV from Vibrio parahaemolyticus) and their cross reactivity in both human and mouse serum samples demonstrating the inability of this system to determine accurately the bacterium elicited antigenicity in human samples. The manuscript is also suffering with the complete lack of standard or control samples in entire experiments and that raised serious question against reproducibility of work.

Authors must include the closely related species and or strains of all five bacteria used in this study to validate it further.

[Response]

We understand some of the concerns raised by the reviewer. There is no standard human anti-V-antigen IgG except for human monoclonal anti-PcrV IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the anti-PcrV monoclonal standard [ref 32], the optical density measured under a consistent condition with the same secondary antibody was used to evaluate the titers. We do not agree with the reviewer’s suggestion to use closely related species or strains of all five bacteria. We used five recombinant V-antigens from five different species. Among the same species, variation in the primary sequences of V-antigens is quite low. Thus, there is no reason to include closely related species or strains. However, in an additional experiment, we included P. aeruginosa OprF as an irrelevant non-V-antigen protein that has no similarity with the tested V-antigens in terms of primary sequences. As a result, in the inhibition ELISA using OprF, preincubation with OprF did not affect the optical density measured for anti-V-antigen titers, whereas preincubation with LssV significantly decreased anti-V-antigen titers for ArcV and LssV as evidence of cross-antigenicity (new Fig. 5 Inhibition ELISA).

As the reviewer pointed out, there is no standard sample for titer measurement of anti-LcrV, anti-LssV, anti-VcrV, and anti-AcrV. Except for human monoclonal anti-PcrV IgG mAb 6F5 as a standard to measure the anti-PcrV titer [32], there is no human standard anti-V-antigen IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the mAb 6F5 standard [32], the optical density measured under a consistent condition with the same secondary antibody was used to evaluate the titers. Accordingly, we have added the following text.

Page 14, line 202 (page 13, line 282 of tracked_change_version): Next, the inhibition ELISA was performed to show cross-antigenicity among V-antigens. As an irrelevant non-crossreactive protein, we prepared P. aeruginosa recombinant OprF using the same E.coli-derived recombinant protein construction system. The sequence alignment scores obtained from ClustalW between OprF and V-antigens were 9.8–11.4, whereas those among V-antigens were from 21.3 (between VcrV and AcrV) to 48.3 (between LcrV and LssV). Three human sera diluted 1000× were preincubated with either recombinant LssV or recombinant OprF overnight. Then, preprocessed serum titers against each V-antigen were measured in comparison with the titer levels of the original sera (Fig. 5). As a result, preincubation with OrpF did not affect the specific titer levels. However, preincubation with LssV decreased the titer levels compared with the titer levels of the original sera (*p<0.05 for AcrV, VcrV, and LssV).

Page 9, line 130 (page 9, line 178 of tracked_change_version): Except for human monoclonal anti-PcrV IgG mAb 6F5 as a standard to measure the anti-PcrV titer [32], there is no human anti-V-antigen IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the mAb 6F5 standard [32], the OD measured under a consistent condition with the same secondary antibody was used to evaluate the titers.

Reviewer #2: Major comments:

1) Figure 4A doesn't match to the written part that describe it and actually it is identical to figure 7A. Please correct the figure.

[Response]

We have corrected the error pointed out by the reviewer. In the revised manuscript, Figure 4A is the correct figure.

2) There is no statistical method section in the Materials and Methods part so it is impossible to determine statistical significance, power analysis etc. Please include such a section with the relevant statistical analyses.

[Response]

As suggested, we have added a new section “Statistical analysis” in the Materials and methods.

3) Regarding the heat maps, please provide a valid statistical/numerical information that defines the various colors. I'm sure that this data is provided by the relevant analysis tools. Please include a short description also in the figure legend.

[Response]

We have added numerical information about the color heat maps in figures. We have added the methodology to construct the heat maps in the newly added section “Phylogenetic and cluster analyses“ and the explanation of the color-index in each figure legend.

Minor comments:

1)The sentence in line 273 is not clear.

[Response]

As pointed out by the reviewer, the description of this sentence was unclear. We have rewritten the sentence as follows.

Page 20, line 316 (page 20, line 525 of tracked_change_version): Regarding the blocking antibody recognizing the three-dimensional conformational epitopes and not the primary amino acid sequences, not only the serum titer levels, but also the specific components that bind to the specific blocking regions in V-antigen molecules may be important to prevent the pathogenesis associated with TTSS virulence.

2)Line 30; a dot is missing after the 0.7

[Response]

The error has been corrected.

3)Line 117; please correct the 0.0M to a valid value.

It has been changed to “0.05 M”.

4)Line 119; please correct the 200mL to 200μL

[Response]

It has been changed to “200 μL”.

5)Line 121; please correct the 100mL/well to 100μL/well

[Response]

It has been changed to “100 μL”.

6)line 125; please correct the 100mL/well to 100μL/well

[Response]

It has been changed to “100 μL”.

7)line 224; please correct "..carboxyl domains.." to carboxyl-terminal domains

[Response]

The error has been corrected.

Reviewer #3: This paper by Kinoshita et. al. looks into the antibody response to various bacterial V-antigens in a cohort of healthy adults. They find that many people have various antibody responses to the V-antigens and that many of these antibodies may cross-react. The data is sound and the authors are careful not to overstate their findings. I do have a few comments however;

1. It is not made clear what percent of people have reactive antibodies to each protein. This could be a percent of patients over a specified cutoff. You already have both ‘red’ and ‘orange’ cutoffs so these could be used.

[Response]

On the basis of our previous results published in ref 18, we have added our speculation about the sufficient levels at which we can anticipate the protective effects because it is difficult to determine the cut-off levels.

Page 24, line 398 (page 24, line 640 of tracked_change_version): In this study, among the 198 volunteer-derived sera [32], the top 10 high titer sera against PcrV (greater than or equivalent to 0.5 at OD 450 nm) were used. Although we have human monoclonal IgG specific against PcrV as a standard for anti-PcrV measurement, no such human standard IgG against the other four V-antigens are available to date. It is difficult at this time to clearly evaluate where the level of protection is sufficient, but as many as 5% of the volunteers showed protective levels in our previous study [18], and the cross-antigenicity among the five V-antigens probably has a certain level of clinical significance in human immunity.

2. Both the correlations in Figure 3 and the mice vaccination studies strongly suggest the presence of some cross-reactive antibodies in the human sera. This would be fairly easy to prove in the human sera by absorption experiments. Take a human sera with responses to multiple proteins- adsorb against the highest reacting protein and determine if this also removes response to the other V-antigens.

[Response]

As the reviewer suggested, we have performed the additional experiment. We conducted an inhibition ELISA (sometimes called a competitive ELISA) to distinguish the levels of specific binding from the levels due to non-specific protein bindings. For high titer sera, we preincubated with either recombinant OprF (a Pseudomonas aeuriginosa surface antigen protein that is irrelevant to the V-antigen) or recombinant LssV and then measured the specific anti-V-antigen titers. The results are shown in new Fig. 5.

Page 14, line 202 (page 13, line282 of tracked_change_version): Next, the inhibition ELISA was performed to show cross-antigenicity among V-antigens. As an irrelevant non-crossreactive protein, we prepared P. aeruginosa recombinant OprF using the same E.coli-derived recombinant protein construction system. The sequence alignment scores obtained from ClustalW between OprF and V-antigens were 9.8–11.4, whereas those among V-antigens were from 21.3 (between VcrV and AcrV) to 48.3 (between LcrV and LssV). Three human sera diluted 1000× were preincubated with either recombinant LssV or recombinant OprF overnight. Then, preprocessed serum titers against each V-antigen were measured in comparison with the titer levels of the original sera (Fig. 5). As a result, preincubation with OrpF did not affect the specific titer levels. However, preincubation with LssV decreased the titer levels compared with the titer levels of the original sera (*p<0.05 for AcrV, VcrV, and LssV).

3. Figure 5 would be easier to interpret if it was a graph or heat map of the response levels instead of a photo of the plate. I can’t tell by eye if one well is more blue than another.

[Response]

In accordance with the reviewer comment, we have omitted the previous Fig. 5.

4. Lines 244-248 the authors state that the amino-acid sequences phylogeny in Fig 7 do not match the serum trees in Fig 4A. To my eye they do match pretty well especially 7A and 7B. In both LssV is closest to LcrV and VcrV is furthest away. Can the authors clarify their statement?

[Response]

We apologize for our error. In Fig 4A, we mistakenly used Fig. 7A. We have corrected the error.

Comments

In this manuscript, Kinoshita et al tried to establish serum anti-V antibody titer as method for the epidemiological investigations on various pathogenic Gram-negative bacteria. Authors developed an enzyme-linked immuno-sorbent assay to measure titers against each V antigen in the sera collected from 186 adult volunteers. However, the manuscript is identified with a rather confusing experimental approaches for epidemiological investigation of Gram-negative bacterial pathogens. The application of purified V antigens (PcrV from Pseudomonas aeruginosa, LcrV from Yersinia, LssV from Photorhabdus luminescens, AcrV from Aeromonas salmonicida and VcrV from Vibrio parahaemolyticus) and their cross reactivity in both human and mouse serum samples demonstrating the inability of this system to determine accurately the bacterium elicited antigenicity in human samples. The manuscript is also suffering with the complete lack of standard or control sample in entire experiments and that raised serious question against reproducibility of work.

[Response]

As the reviewer pointed out, there is no standard sample for titer measurement of anti-LcrV, anti-LssV, anti-VcrV, and anti-AcrV. Except for human monoclonal anti-PcrV IgG mAb 6F5 as a standard to measure the anti-PcrV titer [32], there is no human standard anti-V-antigen IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the mAb 6F5 standard [32], the optical density measured under a consistent condition with the same secondary antibody was used to evaluate the titers. We added the description about it in Page 9, line 130, as follows:

“Except for human monoclonal anti-PcrV IgG mAb 6F5 as a standard to measure the anti-PcrV titer [32], there is no human anti-V-antigen IgG. Therefore, after optimization of the ELISA system for anti-PcrV titers using the mAb 6F5 standard [32], the OD measured under a consistent condition with the same secondary antibody was used to evaluate the titers.”

Accordingly, we have added the following text. In addition, to confirm the cross-antigenicity, we added an additional experiment (inhibition ELISA with irrelevant protein OprF), and demonstrated the result in the new Fig 5.

Submitted filename: ResponseToComments.docx

Click here for additional data file.

29 Jan 2020

PONE-D-19-20754R1

Epidemiological survey of serum titers from adults against various Gram-negative bacterial V-antigens

PLOS ONE

Dear Dr. Sawa,

Thank you for submitting your  revised manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Before accepting it for publication I would like to ask you to carefully review and clarify/fix the remaining small issues (scientific and grammatical) as noted by the reviewers.

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Katerina Kourentzi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Thank you for your revised manuscript!

Before accepting it for publication I would like to ask you to carefully review and clarify/fix the remaining small issues (scientific and grammatical) as noted by the reviewers.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have modified the manuscript as per the comments. However, revised manuscript is still having few grammatical mistakes, which need to be addressed before final submission.

Reviewer #2: I acknowledge the authors for addressing my remarks. Few points that still need attention.

In lines 163, 165 and 166 of the revised manuscript there are empty parentheses that either need to be filled or should be deleted.

In figure 4B the order of the V antigen of the 5 species on the two axes is not the same. Is there a reason for that? If the answer is no please correct the order.

Reviewer #3: As stated above all my comments have been addressed sufficiently. The new Figure 5 improves the manuscipt especially.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Feb 2020

Review Comments to the Author

Reviewer #1: Authors have modified the manuscript as per the comments. However, revised manuscript is still having few grammatical mistakes, which need to be addressed before final submission.

[Response to the comment]

We fixed the following grammatical errors.

Page 4, line 50: the virulence associated -> the virulence-associated

Page 6, line 74: correlated and -> correlated, and

Page 6, line 87: multicloning site -> multiple cloning site

Page 8, line 108: March 2013 participated -> March 2013, participated

Page 9, line 128: a typo error: inhibiton -> inhibition

Page 11, line 153: by ELISAs as described -> by ELISAs, as described

Page 11, line 161: method and rooted trees -> method, and rooted trees

Page 12, line 176: regression analysis -> a regression analysis

Page 12, line 180: as considered statistically significant. -> considered statistically significant.

Page 13, line 204: coefficient 0.84 -> coefficient of 0.84

Page 14, line 226: unique profile -> a unique profile

Page 15, line 235: method -> method,

Page 15, line 240: method -> method,

Page 17, line 278: additional sequence -> an additional sequence

Page 22, line 357: a challenge infection -> challenge infection

Page 22, line 369: shrimp and -> shrimp, and

Page 23, line 380: Luminescens -> luminescens

Page 25, line 414: the type III secretory apparatus -> type III secretory apparatus

Page 25, line 416: better understanding -> a better understanding

Page 26, line 431: Science and -> Science, and

Page 26, line 433: PhD, -> Ph.D.,

Reviewer #2: I acknowledge the authors for addressing my remarks. Few points that still need attention.

In lines 163, 165 and 166 of the revised manuscript there are empty parentheses that either need to be filled or should be deleted.

[Response to the comment]

Page 11, line 162- 165 fixed (Actually, there are not empty parentheses, but the R function(). However, we eliminated the parentheses to avoid the confusion)

In figure 4B the order of the V antigen of the 5 species on the two axes is not the same. Is there a reason for that? If the answer is no please correct the order.

[Response to the comment]

We fixed the error. Now, we aligned them in a correct order.

Reviewer #3: As stated above all my comments have been addressed sufficiently. The new Figure 5 improves the manuscript especially.

[Response to the comment]

We thank you very much for your good advice!

Submitted filename: Response to Reviewers_2nd.docx

Click here for additional data file.

25 Feb 2020

Epidemiological survey of serum titers from adults against various Gram-negative bacterial V-antigens

PONE-D-19-20754R2

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Additional Editor Comments (optional):

Reviewers' comments:

27 Feb 2020

PONE-D-19-20754R2

Epidemiological survey of serum titers from adults against various Gram-negative bacterial V-antigens

Dear Dr. Sawa:

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PLOS ONE

Table 1

Gene sources, primer sets for V-antigen and OprF gene cloning, and characteristics of the recombinant V-antigens and OprF used in this study.

Gene	Gene source	Restriction enzyme site for expression vector	Cloning PCR primers*	Coding region size (bp)	Protein	Amino -acids	MW (kDa)	Ref
pcrV	Pseudomonas aeruginosa PA103	BamHIHindIII	5'-CGGGATCCATGGAAGTCAGAAACTTTAA-3'5'-AAGCTTCTAGATCGCGCTGAGAATGT-3'	927	PcrV	306	33.8	[34–36]
lcrV	Yersinia pestis pCD1 plasmid	BamHIHindIII	5'-CGGGATCCATGATTAGAGCCTACGAACAAAACCCACAA-3'5'-AAGCTTTCATTTACCAGACGTGTCATCTAGCAGACG-3'	1,023	LcrV	338	38.6	[9]
acrV	Plasmid JF2267 of Aeromonas salmonicida subsp. Salmonicida,	SphISalI	5’-GCATGCATGAGCACAATCCCTGACTAC-3’5’-GTCGACTCAAATTGCGCCAAGAATGTCG-3’	1,134	AcrV	375	41.6	14
vcrV	Vibrio parahaemolyticus, ATCC 17802D-5	BamHIPstI	5’-CGGGATCCATGACGGATATGACAACAAC-3’5’-CTGCAGTTAAATGGCTCGTAGGATTTCTTG-3’	1,866	VcrV	619	68.3	-
lssV	Photorhabdus luminescens subsp, luminescens, ATCC 29999	BamHIHindIII	5’-GGATCCATGGAAATAGGCCATATCAAA-3’5’-AAGCTTTTAAATTGCGCCGAGAATAT-3’	1,020	LssV	337	38.3	-
oprF	Pseudomonas aeruginosa PAO1	BamHIHindIII	5’-CGGGATCCATGAAACTGAAGAACACCTTAG -3’5’-AAGCTTTTACTTGGCTTC AGCTTCTAC-3’	1,053	OprF	362	39.0	-

*Underlines indicate restriction enzyme sites