Large scale controlled Fab exchange GMP process to prepare bispecific antibodies.

Objective: Bispecific antibodies (BsAbs) have demonstrated significant therapeutic impacts for the treatment of a broad spectrum of diseases that include oncology, auto-immune, and infectious diseases. However, the large-scale production of clinical batches of bispecific antibodies still has many challenges that include having low yield, poor stability, and laborious downstream purification processes. To address such challenges, we describe the optimization of the controlled Fab arm exchange (cFAE) process for the efficient generation of BsAbs. Methods: The process optimization of a large-scale good manufacturing practice (GMP) cFAE strategy to prepare BsAbs was based on screening the parameters of temperature, reduction, oxidation, and buffer exchange. We include critical quality standards for the reducing agent cysteamine hydrochloride. Results: This large-scale production protocol enabled the generation of bispecific antibodies with >90% exchange yield and at >95% purity. The subsequent downstream processing could use typical mAb procedures. Furthermore, we demonstrated that the bispecific generation protocol can be scaled up to ∼60 L reaction scale using parental monoclonal antibodies that were expressed in a 200 L bioreactor. Conclusion: We presented a robust development strategy for the cFAE process that can be used for a larger scale GMP BsAb production.

The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans.

In Streptococcus mutans, we find that the histidine kinase WalK possesses the longest C-terminal tail (CTT) among all 14 TCSs, and this tail plays a key role in the interaction of WalK with its response regulator WalR. We demonstrate that the intrinsically disordered CTT is characterized by a conserved tryptophan residue surrounded by acidic amino acids. Mutation in the tryptophan not only disrupts the stable interaction, but also impairs the efficient phosphotransferase and phosphatase activities of WalRK. In addition, the tryptophan is important for WalK to compete with DNA containing a WalR binding motif for the WalR interaction. We further show that the tryptophan is important for in vivo transcriptional regulation and bacterial biofilm formation by S. mutans. Moreover, Staphylococcus aureus WalK also has a characteristic CTT, albeit relatively shorter, with a conserved W-acidic motif, that is required for the WalRK interaction in vitro. Together, these data reveal that the W-acidic motif of WalK is indispensable for its interaction with WalR, thereby playing a key role in the WalRK-dependent signal transduction, transcriptional regulation and biofilm formation.

Structural insights into the signal transduction mechanism of the K+-sensing two-component system KdpDE.

Two-component systems (TCSs), which consist of a histidine kinase (HK) sensor and a response regulator (RR), are important for bacteria to quickly sense and respond to various environmental signals. HKs and RRs typically function as a cognate pair, interacting only with one another to transduce signaling. Precise signal transduction in a TCS depends on the specific interactions between the receiver domain (RD) of the RR and the dimerization and histidine phosphorylation domain (DHp) of the HK. Here, we determined the complex structure of KdpDE, a TCS consisting of the HK KdpD and the RR KdpE, which is responsible for K+ homeostasis. Both the RD and the DNA binding domain (DBD) of KdpE interacted with KdpD. Although the RD of KdpE and the DHp of KdpD contributed to binding specificity, the DBD mediated a distinct interaction with the catalytic ATP-binding (CA) domain of KdpD that was indispensable for KdpDE-mediated signal transduction. Moreover, the DBD-CA interface largely overlapped with that of the DBD-DNA complex, leading to competition between KdpD and its target promoter in a KdpE phosphorylation-dependent manner. In addition, the extended C-terminal tail of the CA domain was critical for stabilizing the interaction with KdpDE and for signal transduction. Together, these data provide a molecular basis for specific KdpD and KdpE interactions that play key roles in efficient signal transduction and transcriptional regulation by this TCS.

Molecular and biochemical characterization of Eimeria tenella hexokinase.

Hexokinase (HK) is one of the key enzymes in the glycolytic pathway that catalyzes the phosphorylation of glucose. In the present study, we cloned the HK gene from the coccidian Eimeria tenella (EtHK), expressed EtHK as a His-tagged fusion protein, and characterized its primary biochemical features. Mutagenesis confirmed that residues S159, N216, and D217 are essential or important to the EtHK catalytic activity. EtHK exhibited high affinity for D-glucose (Km = 0.67 to 0.79 mM), but was also able to utilize 2-deoxy-D-glucose (Km = 5.66 mM), D-fructose (Km = 13.76 mM), and D-mannose (Km = 25.41 mM). We also observed that quercetin and mangiferin could inhibit the EtHK enzyme activity (IC50 values = 6.52 and 85.82 µM, respectively). Among the two inhibitors, mangiferin also inhibited the growth of E. tenella in vitro (MIC50 = 0.12 µM). These observations suggest that EtHK may be explored as potential drug target, and mangiferin and its analogs may be explored for developing anti-coccidial therapeutics.

The complete mitochondrial genome of Tetrancistrum nebulosi (Monogenea: Ancyrocephalidae).

The first complete mitochondrial (mt) genome of Ancyrocephalidae is reported herein. The mt genome of Tetrancistrum nebulosi was 13,392 bp in length containing 12 protein-coding genes (lacking atp8), 22 tRNA genes and 2 rRNA genes. The longest non-coding region was located between nad5 and trnG, and the A + T content was 72.4%. All tRNAs had the typical clover-leaf secondary structure except for trnS1((AGN)), trnR, trnF and trnQ. The rrnL and rrnS subunits were separated by trnC, as documented in the monopisthocotylean groups (Benedenia and Gyrodactylus species), while they were adjacent to each other in the polyopisthocotylean species (Microcotyle sebastis, Polylabris halichoeres and Pseudochauhanea macrorchis).

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

OBJECTIVES: To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. DESIGN AND METHODS: We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. RESULTS: This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. CONCLUSIONS: An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

Novel monoclonal antibodies against Plasmodium falciparum histidine-rich protein 2: development and application in rapid diagnostic tests of malaria in hyperendemic regions of China and Myanmar.

BACKGROUND: Malaria presents a considerable threat to public health. Histidine-rich protein 2 (HRP 2) is the major protein released into human blood upon infection by Plasmodium falciparum. In this study, we aimed to evaluate the immunogenicity of HRP 2 exon II and the efficacy of novel monoclonal antibodies (mAbs) against HRP 2 for Point-of-Care Test (POCT). METHODS: The recombinant protein was expressed in soluble form in E. coli and used to immunize mice for mAb production. Two IgG1 mAbs (1A5 and 1C10) with high affinity, specificity and sensitivity for both native and recombinant HRP 2 were selected after fusion of mouse spleen with myeloma cells. The affinity constant of 1A5 and 1C10 were 7.15 and 4.91 × 10-7 L/mol, respectively. Subsequently, an immunochromatograhic assay was used for screening of clinical samples in endemic regions of China and Myanmar. RESULTS: The immunochromatographic test retrospectively showed an overall sensitivity of 99.07%, and specificity of 100%. Sensitivity at parasite densities < 200, 200-2000, and > 2000 parasites/µL was 87.5, 98.7, and 100%, respectively. CONCLUSIONS: These results suggest that HRP 2 exon II contains immunogenic sites similar to those of the native antigen and can be used for the development of mAbs suitable for malaria diagnosis in endemic communities.

Development and performance evaluation of a novel immunofluorescence chromatographic assay for histidine-rich protein 2 of Plasmodium falciparum.

BACKGROUND: The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. METHODS: A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. RESULTS: The cut-off level of detection of the kit was 25 parasites/µl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. CONCLUSIONS: A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.

Production, purification and application of polysaccharide-based bioflocculant by Paenibacillus mucilaginosus.

The production and purification of polysaccharide-based bioflocculants (PSBs) by Paenibacillus mucilaginosus GIM1.16 in metal ion-supplemented medium and basal medium were evaluated. Three purified PSB1-1, PSB2-1 and PSB3-1 possessed different monosaccharide composition and their molecular weights were 2.53 × 10(6), 7.77 × 10(6) and 13.2 × 10(6)Da, respectively. FT-IR spectrometry indicated the presence of hydroxyl, carboxyl and phosphate groups in the three samples. Scanning electron microscopy showed that they had linear structure. The potential of these PSBs on wastewater treatment was evaluated. Among them, PSB1-1 exhibited the best performance, as it had high flocculating activities (above 94%) at 0.5-4 mg/L and could achieve high flocculating activities (above 97%) in the kaolin suspensions of pH 3-9. PSB1-1 was the key factor that might explain the enhanced flocculating activity of the supernatant from metal ion-supplemented medium. The performance of PSB1-1 on industrial wastewater was also satisfactory. PSB1-1 might be a good candidate as bioflocculant.

The complete mitochondrial genome of Neobenedenia melleni (Platyhelminthes: Monogenea): mitochondrial gene content, arrangement and composition compared with two Benedenia species.

The complete mitochondrial (mt) genome sequences of Neobenedenia melleni were determined and compared with those of Benedenia seriolae and B. hoshinai. This circular genome comprises 13,270 bp and includes all 36 typical mt genes found in flatworms. Total AT content of N. melleni is 75.9 %. ATG is the most common start codon, while nad4L is initiated by GTG. All protein-coding genes are predicted to terminate with TAG and TAA. N. melleni has the trnR with a TCG anticodon, which is the same to B. seriolae but different from B. hoshinai (ACG). The mt gene arrangement of N. melleni is similar to that of B. seriolae and B. hoshinai with the exception of three translocations (trnF, trnT and trnG). The overlapped region between nad4L and nad4 was found in the N. melleni mt genome, which was also reported for the published Gyrodactylus species, but it was not found in those of B. seriolae and B. hoshinai, which are non-coding regions instead. The present study provides useful molecular characters for species or strain identification and systematic studies of this parasite.

Development of rapid immunochromatographic test for hemagglutinin antigen of H7 subtype in patients infected with novel avian influenza A (H7N9) virus.

BACKGROUND: Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. METHODOLOGY/PRINCIPAL FINDINGS: We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. CONCLUSIONS/SIGNIFICANCE: Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic.

Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris.

A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained >90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from ß-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.

Molecular cloning, recombinant expression and antibacterial activity analysis of hepcidin from Simensis crocodile (Crocodylus siamensis).

Hepcidin, a cysteine-rich cationic antibacterial peptide, plays an important role in human defense against pathogen infection. However, its role in reptile immune response and whether it is involved in antibacterial immune have not yet been proven. In order to study the antibacterial activity of Crocodylus siamensis hepcidin (Cshepc), a common reptile which lives in topic region of Southeast Asia, a cDNA sequence of Cshepc was cloned, which included an open reading frame (ORF) of 300 bp encoding a 99 amino acid preprohepcidin. Cshepc has eight cysteines formed four conserved disulfide bridges, similarly to that of human's. Sequence analysis showed that Cshepc mature peptide was more conserved than that of preprohepcidin. Tissue expression analysis indicated that Cshepc transcripts were highly expressed in the liver, muscle and heart of C. siamensis. Recombinant expressed hepcidin could significantly inhibit the growth of the Gram-negative bacteria Escherichia coli and Aeromonas sobria as well as the Gram-positive bacterium Staphylococcus aureus, and Bacillus subtilis in vitro, suggesting that Cshepc, like human hepcidin could play a role in the antibacterial function in hosts innate immune response.

The complete mitochondrial genome of Pseudochauhanea macrorchis (Monogenea: Chauhaneidae) revealed a highly repetitive region and a gene rearrangement hot spot in Polyopisthocotylea.

The complete mitochondrial genome of Pseudochauhanea macrorchis was determined and compared with other monogenean mitochondrial genomes from GenBank. The circular genome was 15,031 bp in length and encoded 36 genes (12 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs) typically found in flatworms. Structures of the mitochondrial genome were mostly concordant with that known for Microcotyle sebastis and Polylabris halichoeres, but also contained two noted features-a gene rearrangement hot spot and the highly repetitive region (HRR) in major non-coding region (NCR). The gene rearrangement hot spot located between the cox3 and nad5 genes, including a cluster of tRNA genes, nad6 gene and one major NCR. The HRR seemed to be a unique feature of the polyopisthocotylean mitochondrial genomes. In conclusion, the present study provided new molecular data for future studies of the comparative mitochondrial genomics and also served as a resource of markers for the studies of species populations and monogenean phylogenetics.

Functional characterizations of malonyl-CoA:acyl carrier protein transacylase (MCAT) in Eimeria tenella.

Eimeria tenella, an apicomplexan parasite in chickens, possesses an apicoplast and its associated metabolic pathways including the Type II fatty acid synthesis (FAS II). Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system. In the present study, the entire E. tenella MCAT gene (EtfabD) was cloned and sequenced. Immunolabeling located this protein in the apicoplast organelle in coccidial sporozoites. Functional replacement of the fabD gene with amber mutation of E. coli temperature-sensitive LA2-89 strain by E. tenella EtMCAT demonstrated that EcFabD and EtMCAT perform the same biochemical function. The recombinant EtMCAT protein was expressed and its general biochemical features were also determined. An alkaloid natural product corytuberine (CAS: 517-56-6) could specifically inhibit the EtMCAT activity (IC(50)=16.47µM), but the inhibition of parasite growth in vitro by corytuberine was very weak (the predicted MIC(50)=0.65mM).

The mitochondrial genome of Polylabris halichoeres (Monogenea: Microcotylidae).

This study presents the complete mitochondrial (mt) genome of Polylabris halichoeres, which is the largest mt genome sequenced of monogeneans so far and the second complete sequence after Microcotyle sebastis from the Microcotylidae. It is basically similar to that of M. sebastis, with the exception of a high level of gene rearrangement located between trnC and trnL((UUR)), a translocation of trnM and trnH, as well as a highly repetitive region (HRR) in the large non-coding region (NCR). We also find a series of trnI pseudogenes (ΨI) and one unknown short open reading frame (ORF) in the large NCR. Although the ORF cannot be unambiguously regarded as an atp8 gene, we cannot rule out the possibility that it has other functional importance, but it need further study in the future.

The serum of rabbitfish (Siganus oramin) has antimicrobial activity to some pathogenic organisms and a novel serum L-amino acid oxidase is isolated.

The serum of rabbitfish (Siganus oramin) has been confirmed previously to have killing effect to Cryptocaryon irritans, an important marine ciliate protozoan that causes a disease referred to as "marine white spot disease". Herein, we find the serum of the rabbitfish also shows antibacterial activity against both gram-positive and gram-negative bacteria and has killing effect on two other parasites: Trypanosoma brucei brucei, Ichthyophthirius multifiliis. Results of scanning electron microscopy indicated that after treating with rabbitfish serum, the surface of the Staphylococcus aureus was wrinkled and pores were formed on the surface of Escherichia coli. Serum of the rabbitfish possesses a strong killing effect to Ichthyophthirius multifiliis in vitro, causing a similar effect as to C. irritans. The serum of rabbitfish also showed strong killing effect to T. b. brucei in vitro, with the minimus trypanocidal titre (MTT) only to be 1.5% in 1 h. Results of laser confocal fluorescence microscopy indicated that rabbitfish serum could also induce cell rupture of T. b. brucei. A novel antimicrobial protein (SR-LAAO) was isolated from the serum of rabbitfish by using ultrafiltration, reversed phase high performance liquid chromatography (RP-HPLC) and Native polyacrylamide gel electrophoresis (Native-PAGE). Results of gel overlay assay showed that the protein could act alone to inhibit the growth of S. aureus and E. coli. Results of western blot and automated Edman degradation showed that it was the same as the antiparasitic protein (APP) reported before to have killing effect on C. irritans. Full length cDNA sequence of the SR-LAAO was cloned. BLAST research suggested that the cDNA of SR-LAAO has a close similarity with a number of L-amino acid oxidases (LAAOs) and possesses two conserved motifs that exist in LAAOs. Combined, these results demonstrate that this protein which has antimicrobial activity to some pathogenic organisms was a novel LAAO found in the serum of rabbitfish. Immunohistochemical analysis demonstrated tissue specific expression and localization of SR-LAAO in the spleen, kidney, gill and blood of the rabbitfish, but was not found in other tissues. These results suggest that this protein may contribute considerably to the host non-specific immune defense mechanism to combat microbes of the rabbitfish and has the potency for using in future drug development.

Roles of L5-7 loop in the structure and chaperone function of SsHSP14.1.

The small heat shock protein SsHSP14.1 from the hyper-thermophilic archeaon, Sulfolobus solfataricus (S. solfataricus) was able to protect proteins from thermal aggregation and prevent enzymes from heat induced inactivation. According to the 3D (dimensional) structural model of SsHSP14.1 developed by us before, the region L5-7 (ß5-ß7, 68-82 residues) plays an important role for the oligomerization of SsHSP14.1 and its chaperone function. Here, to validate the findings, an in-depth investigation was conducted of both the wild type SsHSP14.1 and its deletion mutant DEL75-79. With E. coli proteins and bromelain as substrate, the deletion mutant DEL75-79 can protect them from thermo-aggregating as effective as the wild protein. Interestingly, unlike the wild protein, DEL75-79 was unable to prevent bromelain and EcoRI from thermo-inactivating. Results of size exclusion HPLC showed that the oligomerization state was changed in mutant protein. This was in accordance with the changed structure and lower hydrophobicity of DEL75-79. These outcomes proved that the L5-7 loop did play a role for the oligomerizing SsHSP14.1, and that the residues 75-79 were indispensable for its function of prevent enzymes from thermo-inactivating.

MtsB, a hydrophobic membrane protein of Streptococcus iniae, is an effective subunit vaccine candidate.

Streptococcus iniae is a major bacterium that causes invasive disease in cultured fish worldwide. The protection relies mainly on anti-microbial compounds and vaccines, and there is much interest in developing S. iniae vaccine based on conserved protein immunogens. Subcellular localization of protein has important influence on its immunogenicity. The surface and extracellular proteins of pathogenic bacteria can be easily recognized by the infected host compare to intracellular proteins, which are the feasible vaccine development targets. However, a putative hydrophobic membrane protein (designated MtsB) of the ATP-binding cassette (ABC) transporter system was found to be protective against S. iniae HD-1 infection when used as an injection vaccine administered intraperitoneally into tilapia. The MtsB protein is present on the cytoplasmic membrane and is expressed in vivo during Kunming mice infection by S. iniae HD-1. This is believed to be the first report on the use of a hydrophobic membrane protein of the ABC system as an S. iniae subunit vaccine.

A solute-binding protein for iron transport in Streptococcus iniae.

BACKGROUND: Streptococcus iniae (S. iniae) is a major pathogen that causes considerable morbidity and mortality in cultured fish worldwide. The pathogen's ability to adapt to the host affects the extent of infection, hence understanding the mechanisms by which S. iniae overcomes physiological stresses during infection will help to identify potential virulence determinants of streptococcal infection. Grow S. iniae under iron-restricted conditions is one approach for identifying host-specific protein expression. Iron plays an important role in many biological processes but it has low solubility under physiological condition. Many microorganisms have been shown to be able to circumvent this nutritional limitation by forming direct contacts with iron-containing proteins through ATP-binding cassette (ABC) transporters. The ABC transporter superfamilies constitute many different systems that are widespread among living organisms with different functions, such as ligands translocation, mRNA translation, and DNA repair. RESULTS: An ABC transporter system, named as mtsABC (metal transport system) was cloned from S. iniae HD-1, and was found to be involved in heme utilization. mtsABC is cotranscribed by three downstream genes, i.e., mtsA, mtsB, and mtsC. In this study, we cloned the first gene of the mtsABC transporter system (mtsA), and purified the corresponding recombinant protein MtsA. The analysis indicated that MtsA is a putative lipoprotein which binds to heme that can serve as an iron source for the microorganism, and is expressed in vivo during Kunming mice infection by S. iniae HD-1. CONCLUSIONS: This is believed to be the first report on the cloning the ABC transporter lipoprotein from S. iniae genomic DNA. Together, our data suggested that MtsA is associated with heme, and is expressed in vivo during Kunming mice infection by S. iniae HD-1 which indicated that it can be a potential candidate for S. iniae subunit vaccine.