# Construction and Evaluation of RecombinantLactococcus lactis Expressing the vscO Gene as a Safe Live Oral Vaccine against Vibrio alginolyticus

Authors: Shiping Yang; Yuanzhi Wu; Yucong Huang; Shaohong Ma; Guangben Wei; Jichang Jian; Shuanghu Cai
Journal: Aquaculture Research
 (2023)

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## Abstract

Vibrio alginolyticus is responsible for significant economic losses in the fish industry. It is urgent to develop a vaccine against V. alginolyticus. In this study, a recombinant Lactococcus lactis expressing the VscO protein of V. alginolyticus (Lc-pMG36e-vscO) is constructed. The Lc-pMG36e-vscO has good hereditary stability. The immune responses in pearl gentian grouper (Epinephelus fuscoguttatus (♀) × E. lanceolatu (♂)) vaccinated with Lc-pMG36e-vscO are evaluated. The titers of specific antibody in the immunized grouper displayed an upward and then downward tendency during the total experiments and reached the peak value of 9.6 at the fourth week after booster immunization. The expression levels of IL-1β, IL-16, TNF-α, and MHC-1α gene in the Lc-pMG36e-vscO group were significantly upregulated after vaccination, indicating that the Lc-pMG36e-vscO could induce an effective and durable immune response. In vivo challenge of the Lc-pMG36e-vscO-immunized fish with V. alginolyticus showed a relative survival percentage of 68%. These results indicated that Lc-pMG36e-vscO could be promising as a candidate oral vaccine against V. alginolyticus.


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## Body

## 1. Introduction

Vibriosis, caused by several species from the family of Vibrionaceae, negatively affects the various marine animals and impedes the sustainable development of the global aquaculture industry [1]. Vibrio alginolyticus is an important pathogen causing vibriosis in the marine culture. In the past several decades, some marine-cultured animals including Lates calcarifer, Sparus aurata, Dicentrarchus labrax, Litopenaeus vannamei, Marsupenaeus japonicas, and Crassostrea gigas suffered serious production losses due to infection by V. alginolyticus [2–7]. It has been reported that V. alginolyticus also infected humans and caused gastroenteritis, wound infections, and septicemia and even amputations and death [8]. Due to the harmful effect caused by V. alginolyticus, farmers had to combat the disease with some treatment ways, such as antibiotics, green water technique, topical disinfectants, medicinal plants, bacteriophage, probiotics, and vaccines [9]. Antibiotics have been excessively used to combat Vibrio spp. infections in aquaculture. Drug resistance and residues are becoming a public health issue, and more stringent requirements and regulations on the use of antibiotics will be imposed [9, 10]. Compared with antibiotics, the vaccine is more effective and safer for preventing vibriosis [9]. Therefore, it is necessary to develop a suitable vaccine against vibriosis.Currently, most vaccines including bacterins (inactivated causative agents), live attenuated vaccines, DNA vaccines, subunit vaccines, polyvalent vaccines, and monovalent vaccines have been developed and researched, which provide some protection in fish [11]. In the current situation, vaccine antigens to protect fish against vibriosis are usually the virulence factors of Vibrio isolates. The recombinant subunit vaccine of the virulent factor against V. alginolyticus is proven to be environment-friendly as an alternative to antibiotics and gives a long-lasting protective immunity to the fish [11]. The type III secretion system (T3SS) is essential for the pathogenesis of V. alginolyticus. VscO, a main component of T3SS, may play a similar chaperone escort role in pathogenicity and be required for normal secretion of T3SS. VscO protein was proven to have good antigenicity and could induce a high antibody titer to protect against lethal challenge with V. alginolyticus. Those results indicated that VscO protein could be used as a candidate antigen for developing a subunit vaccine [12].Probiotics have been applied in aquaculture for several decades.Lactococcus lactis, an endogenous microbe, could exert growth performance, immune response, and disease resistance in aquaculture animals [13–15]. As an endogenous microbe, L. lactis could adhere to mucosal surfaces of the gut and then colonize, establish, and multiply in the fish gut [13]. The mucosal surface is an important physiological barrier and provides a portal of entry for pathogens [16]. Adequate systemic immune response protecting the host could be induced on the mucosal surface [17]. The application of probiotics delivering specific protective antigens to the mucosal surfaces may be an alternative way. In previous studies, Lactococcus is generally regarded as a safe probiotic and has been used to produce vaccines expressing the antigens, such as OmpAI, flagellin A protein, and OmpC of Aeromonas veronii [16, 18, 19] and immobilization antigen IAG-52X of Ichthyophthirius multifiliis [20]. Antigens expressed by Lactococcus are presented to the immune system in particulate form, which may be less likely to induce oral tolerance than soluble antigens. These studies have indicated that Lactococcus is an appropriate candidate for heterologous antigens delivery to mucosal sites.In the present study,L. lactis is isolated from the intestine of healthy pearl gentian grouper (♀Epinephelus fuscoguttatus×♂E. lanceolatu); vscO gene of V. alginolyticus is transferred into L. lactis for constructing a recombinant L. lactis strain. The natural antigenicity of the recombinant VscO expressed in L. lactis is explored. Furthermore, the potential of the recombinant L. lactis strain as an oral live vaccine is evaluated in pearl gentian grouper administrated orally with the strain.


## 2. Materials and Methods

### 2.1. Ethics Statement

All experimental protocols used in animal experiments were approved by the Animal Care and Use Committee of Guangdong Ocean University.


### 2.2. Bacterial Strains, Plasmids, and Growth Conditions

L. lactis was isolated from the intestine of pearl gentian grouper and cultured in De Man, Rogosa, and Sharpe medium (MRS, Huankai, China) at 30°C. Safety assays revealed that the L. lactis was not virulent for grouper. V. alginolyticus strain HY9901 isolated from the diseased maricultured fish was cultured in tryptic soy broth medium (TSB, Huankai, China) supplemented with 2% NaCl at 28°C [21]. Escherichia coli (DH5α) were cultured in Luria−Bertani medium (LB, Huankai, China) at 37°C. The vector pMG36e containing an encoding erm gene (Erythromycin) was purchased from BioVector NTCC Inc in China. When required, the appropriate antibiotic Erm (10 μg/mL) was added in the medium.


### 2.3. Construction of RecombinantL. lactis Expressing VscO Protein

The vscO gene (462 bp) ofV. alginolyticus was amplified by PCR using the following primers with Xba I and Hind III site underlined (Table 1). The PCR product of vscO and the plasmid pMG36e were cleaved with Xba I and Hind III, respectively. Then, the processed fragment was ligated to pMG36e. Finally, the recombinant pMG36e-vscO plasmid was transformed into L. lactis by electroporation as previously described [22]. The strain containing pMG36e-vscO plasmid was named Lc-pMG36e-vscO. The wild L. lactis and PBS were used as control.Table 1
Primers used in this study.
PrimerNucleotide sequence (5’-3’)Accession no.vscO-RGCT​CTA​GAG​ATG​ATA​GAA​CGT​TTA​TTA​GA (Xba I)JX131326.1vscO-FCCC​AAG​CTT​TTA​GAT​AAT​GTC​GAC​AGT​GCG (Hind III)pMG36e-RGCC​TCC​TCA​TCC​TCT​TCA​TCUniversal primerpMG36e-FAAT​ATC​GTA​GCG​CCG​GGG​TAIL-1β-RACG​CTG​CTG​GAC​CTT​TAT​CGXM_033647455.1IL-1β-FACA​CGG​CTT​TGT​CGT​CTT​TCTNF-α-RCTT​CCG​TCG​CTG​TCC​TCA​TGT​GFJ491411.1TNF-α-FGCC​ACA​GGA​TCT​GGC​GCT​ACT​CMHC-1α-RTCC​ATC​GTG​GTT​GGG​GAT​GAT​CFJ896112.3MHC-1α-FGCC​GCC​ACG​CTA​CAG​GTT​TCT​AIL-16-RTCT​GTT​CTG​CGG​GTT​TAG​CKP025949.1IL-16-FTTC​AGA​TCC​TCC​GTC​CAA​Cβ-actin-RTCA​GGA​TAC​CCC​TCT​TGC​TCTAY510710.2β-actin-FAAATCGCCGCACTGGTTG


### 2.4. Western Blot Analysis

Western blot analysis was applied to detect the expression of VscO protein in the recombinant Lc-pMG36e-vscO as described in the previous study [23]. Briefly, the total protein of Lc-pMG36e-vscO and wild L. lactis was electrophoresed by SDS-PAGE on a 5% stacking gel and 15% separating gel under denaturing conditions. The protein was electrically transferred from the gel to the nitrocellulose membrane using a semidry apparatus (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skim milk and then incubated with mouse anti-VscO serum at a dilution of 1 : 1000 with blocking buffer (previously prepared in our laboratory) and was kept overnight at 4°C; the secondary antibody was peroxidase-conjugated goat anti-mouse IgG (Sigma, USA) used at 1 : 10000 dilutions. Finally, the blots were visualized by chemiluminescence detection with Western ECL substrate (Thermo Scientific) in an Amersham Imager 600 (GE Healthcare, UK).


### 2.5. Hereditary Stability of Lc-pMG36e-vscO

The hereditary stability of Lc-pMG36e-vscO was measured according to previous research [24]. The Lc-pMG36e-vscO was cultured in MRS medium with erythromycin 10 μg/mL, without shaking at 30°C. After 24 h, the plasmid was extracted from the cells and PCR was used to confirm the presence of vscO fragment in strain using specific primers vscO-R/vscO-F and universal primers pMG36e-R/pMG36e-F for vscO gene (Table 1).


### 2.6. Vaccine Preparation, Oral Vaccination, and Sample Collection

The Lc-pMG36e-vscO and wild strain were cultured to the logarithmic stage. The cultures were collected by centrifugation at 8000 r/min at 4°C for 10 min. Then 2.5% sodium alginate was added and mixed evenly with a commercial grouper pellet diet. The prepared diet was dried at room temperature and ventilation and stored at 4°C. The concentration ofL. lactis is 1.0 × 109 CFU/g diet. The PBS blank control group was treated in the same way.Healthy grouper with a mean weight of 50.0 ± 1.0 g was purchased from a fish farm in Donghai Island (Guangdong Province, China). Fish were allowed to acclimatize for two weeks before vaccination. They were randomly divided into three groups with 3 replicates and 30 fishes per replicate and were fed to apparent satiation twice daily (8 : 30 and 16 : 30) for 42 d with a commercial grouper pellet diet. Fish were immunized with the prepared diets from day 1 to day 7. The booster immunizations were administered from day 21 to day 28. The strategy of oral administration of grouper and sampling is shown in Figure1. Four fish in each group were euthanized with 100 μg/mL of tricaine methane-sulfonate (MS-222, Sigma Aldrich) at 7, 14, 21, 28, 35, and 42 d after the immunization to collect blood, spleen, kidney, and hindgut. The tissues were collected and stored in RNAlater (Thermo Fisher, China) at −80°C for RNA extraction. Sera were kept at −80°C for ELISA.Figure 1
Strategy of oral administration of pearl gentian grouper and sampling.


### 2.7. Analysis of Specific Antibody Levels

Antibody titers were determined as described previously [21]. Briefly, 96-well microtiter plates were coated with the purified VscO protein. Fish sera were added to the microplate in twofold serial dilutions (21 to 210). Plates in duplicate were incubated for 1 h under gentle agitation. Rabbit anti-grouper IgM was used as a secondary antibody and peroxidase-conjugated goat anti-rabbit IgG (Sigma) as tertiary antibodies. Enzyme substrate (o-phenylenediamine, Sigma) was added to the plates, and these were incubated for 10–15 min. The enzymatic reaction was stopped by adding 3 M HCl, and optical density (OD) was determined at 492 nm using an ELISA reader. Each plate included two controls, namely, wells with all antibodies and substrates except sample material (primary antibodies) as negative controls (blanks) and sera obtained from native fish. Values greater than twice the (blank) background absorbance were considered to be positive. ELISA was validated by means of the OD readings obtained from native sera, which were the same in the different plates. The experiments were replicated in triplicate.


### 2.8. Detection of Immune-Related Genes

The total RNA of examined tissues including the spleen, kidney, and hindgut was extracted by Trizol reagent (Invitrogen). Genomic DNA was removed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara). RNA concentration was measured with a spectrophotometer (NanoDrop Technologies). RNA was reverse transcribed into cDNA using the One-Step RT-PCR Kit. The expression levels of immune-related genes (IL-1β, IL-16, TNF-α, and MHC-1α) were detected by real-time quantitative PCR (qRT-PCR) in SYBR∗ Green qPCR SuperMix Kit (Trans-Gen, China). The final reaction volume of 10 μL contained 0.5 μL cDNA, 5 μL of 2 × SYBR Green qPCR SuperMix, 0.5 μL of each primer (10 μM), and 3.5 μL ddH2O. The reaction was performed under the following conditions: 95°C, 2 min; 95°C, 30s; 57°C, 30 s, 72°C, 20 s (40 cycles). A melting curve analysis was performed to access the amplification of specific products. The β-actin gene was used as an endogenous control. The expressions of immune-related genes were normalized to β-actin using the 2−ΔΔCt method. All samples were replicated in triplicate. The primers are shown in Table 1.


### 2.9. Challenge Experiments

At 42 d postimmunization, all the vaccinated fish were injected intraperitoneally with 200μL of 1 × 106 CFU/g fish body weight (5 LD50 doses) of V. alginolyticus strain HY9901. Fish injected with 200 μL PBS were used as the negative control group. The fish challenged with V. alginolyticus were monitored for 15 d, and the survival rate was analyzed in all the group post challenge. The experiments were replicated in triplicate.


### 2.10. Statistical Analysis

Statistical analysis was performed using SPSS 19.0 software. For multiple comparisons, one-way ANOVA is followed by Tukey’s test. Data are expressed as mean ± SD. In all cases, significant differences were considered atP < 0.05.


## 2.1. Ethics Statement

All experimental protocols used in animal experiments were approved by the Animal Care and Use Committee of Guangdong Ocean University.


## 2.2. Bacterial Strains, Plasmids, and Growth Conditions

L. lactis was isolated from the intestine of pearl gentian grouper and cultured in De Man, Rogosa, and Sharpe medium (MRS, Huankai, China) at 30°C. Safety assays revealed that the L. lactis was not virulent for grouper. V. alginolyticus strain HY9901 isolated from the diseased maricultured fish was cultured in tryptic soy broth medium (TSB, Huankai, China) supplemented with 2% NaCl at 28°C [21]. Escherichia coli (DH5α) were cultured in Luria−Bertani medium (LB, Huankai, China) at 37°C. The vector pMG36e containing an encoding erm gene (Erythromycin) was purchased from BioVector NTCC Inc in China. When required, the appropriate antibiotic Erm (10 μg/mL) was added in the medium.


## 2.3. Construction of RecombinantL. lactis Expressing VscO Protein

The vscO gene (462 bp) ofV. alginolyticus was amplified by PCR using the following primers with Xba I and Hind III site underlined (Table 1). The PCR product of vscO and the plasmid pMG36e were cleaved with Xba I and Hind III, respectively. Then, the processed fragment was ligated to pMG36e. Finally, the recombinant pMG36e-vscO plasmid was transformed into L. lactis by electroporation as previously described [22]. The strain containing pMG36e-vscO plasmid was named Lc-pMG36e-vscO. The wild L. lactis and PBS were used as control.Table 1
Primers used in this study.
PrimerNucleotide sequence (5’-3’)Accession no.vscO-RGCT​CTA​GAG​ATG​ATA​GAA​CGT​TTA​TTA​GA (Xba I)JX131326.1vscO-FCCC​AAG​CTT​TTA​GAT​AAT​GTC​GAC​AGT​GCG (Hind III)pMG36e-RGCC​TCC​TCA​TCC​TCT​TCA​TCUniversal primerpMG36e-FAAT​ATC​GTA​GCG​CCG​GGG​TAIL-1β-RACG​CTG​CTG​GAC​CTT​TAT​CGXM_033647455.1IL-1β-FACA​CGG​CTT​TGT​CGT​CTT​TCTNF-α-RCTT​CCG​TCG​CTG​TCC​TCA​TGT​GFJ491411.1TNF-α-FGCC​ACA​GGA​TCT​GGC​GCT​ACT​CMHC-1α-RTCC​ATC​GTG​GTT​GGG​GAT​GAT​CFJ896112.3MHC-1α-FGCC​GCC​ACG​CTA​CAG​GTT​TCT​AIL-16-RTCT​GTT​CTG​CGG​GTT​TAG​CKP025949.1IL-16-FTTC​AGA​TCC​TCC​GTC​CAA​Cβ-actin-RTCA​GGA​TAC​CCC​TCT​TGC​TCTAY510710.2β-actin-FAAATCGCCGCACTGGTTG


## 2.4. Western Blot Analysis

Western blot analysis was applied to detect the expression of VscO protein in the recombinant Lc-pMG36e-vscO as described in the previous study [23]. Briefly, the total protein of Lc-pMG36e-vscO and wild L. lactis was electrophoresed by SDS-PAGE on a 5% stacking gel and 15% separating gel under denaturing conditions. The protein was electrically transferred from the gel to the nitrocellulose membrane using a semidry apparatus (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skim milk and then incubated with mouse anti-VscO serum at a dilution of 1 : 1000 with blocking buffer (previously prepared in our laboratory) and was kept overnight at 4°C; the secondary antibody was peroxidase-conjugated goat anti-mouse IgG (Sigma, USA) used at 1 : 10000 dilutions. Finally, the blots were visualized by chemiluminescence detection with Western ECL substrate (Thermo Scientific) in an Amersham Imager 600 (GE Healthcare, UK).


## 2.5. Hereditary Stability of Lc-pMG36e-vscO

The hereditary stability of Lc-pMG36e-vscO was measured according to previous research [24]. The Lc-pMG36e-vscO was cultured in MRS medium with erythromycin 10 μg/mL, without shaking at 30°C. After 24 h, the plasmid was extracted from the cells and PCR was used to confirm the presence of vscO fragment in strain using specific primers vscO-R/vscO-F and universal primers pMG36e-R/pMG36e-F for vscO gene (Table 1).


## 2.6. Vaccine Preparation, Oral Vaccination, and Sample Collection

The Lc-pMG36e-vscO and wild strain were cultured to the logarithmic stage. The cultures were collected by centrifugation at 8000 r/min at 4°C for 10 min. Then 2.5% sodium alginate was added and mixed evenly with a commercial grouper pellet diet. The prepared diet was dried at room temperature and ventilation and stored at 4°C. The concentration ofL. lactis is 1.0 × 109 CFU/g diet. The PBS blank control group was treated in the same way.Healthy grouper with a mean weight of 50.0 ± 1.0 g was purchased from a fish farm in Donghai Island (Guangdong Province, China). Fish were allowed to acclimatize for two weeks before vaccination. They were randomly divided into three groups with 3 replicates and 30 fishes per replicate and were fed to apparent satiation twice daily (8 : 30 and 16 : 30) for 42 d with a commercial grouper pellet diet. Fish were immunized with the prepared diets from day 1 to day 7. The booster immunizations were administered from day 21 to day 28. The strategy of oral administration of grouper and sampling is shown in Figure1. Four fish in each group were euthanized with 100 μg/mL of tricaine methane-sulfonate (MS-222, Sigma Aldrich) at 7, 14, 21, 28, 35, and 42 d after the immunization to collect blood, spleen, kidney, and hindgut. The tissues were collected and stored in RNAlater (Thermo Fisher, China) at −80°C for RNA extraction. Sera were kept at −80°C for ELISA.Figure 1
Strategy of oral administration of pearl gentian grouper and sampling.


## 2.7. Analysis of Specific Antibody Levels

Antibody titers were determined as described previously [21]. Briefly, 96-well microtiter plates were coated with the purified VscO protein. Fish sera were added to the microplate in twofold serial dilutions (21 to 210). Plates in duplicate were incubated for 1 h under gentle agitation. Rabbit anti-grouper IgM was used as a secondary antibody and peroxidase-conjugated goat anti-rabbit IgG (Sigma) as tertiary antibodies. Enzyme substrate (o-phenylenediamine, Sigma) was added to the plates, and these were incubated for 10–15 min. The enzymatic reaction was stopped by adding 3 M HCl, and optical density (OD) was determined at 492 nm using an ELISA reader. Each plate included two controls, namely, wells with all antibodies and substrates except sample material (primary antibodies) as negative controls (blanks) and sera obtained from native fish. Values greater than twice the (blank) background absorbance were considered to be positive. ELISA was validated by means of the OD readings obtained from native sera, which were the same in the different plates. The experiments were replicated in triplicate.


## 2.8. Detection of Immune-Related Genes

The total RNA of examined tissues including the spleen, kidney, and hindgut was extracted by Trizol reagent (Invitrogen). Genomic DNA was removed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara). RNA concentration was measured with a spectrophotometer (NanoDrop Technologies). RNA was reverse transcribed into cDNA using the One-Step RT-PCR Kit. The expression levels of immune-related genes (IL-1β, IL-16, TNF-α, and MHC-1α) were detected by real-time quantitative PCR (qRT-PCR) in SYBR∗ Green qPCR SuperMix Kit (Trans-Gen, China). The final reaction volume of 10 μL contained 0.5 μL cDNA, 5 μL of 2 × SYBR Green qPCR SuperMix, 0.5 μL of each primer (10 μM), and 3.5 μL ddH2O. The reaction was performed under the following conditions: 95°C, 2 min; 95°C, 30s; 57°C, 30 s, 72°C, 20 s (40 cycles). A melting curve analysis was performed to access the amplification of specific products. The β-actin gene was used as an endogenous control. The expressions of immune-related genes were normalized to β-actin using the 2−ΔΔCt method. All samples were replicated in triplicate. The primers are shown in Table 1.


## 2.9. Challenge Experiments

At 42 d postimmunization, all the vaccinated fish were injected intraperitoneally with 200μL of 1 × 106 CFU/g fish body weight (5 LD50 doses) of V. alginolyticus strain HY9901. Fish injected with 200 μL PBS were used as the negative control group. The fish challenged with V. alginolyticus were monitored for 15 d, and the survival rate was analyzed in all the group post challenge. The experiments were replicated in triplicate.


## 2.10. Statistical Analysis

Statistical analysis was performed using SPSS 19.0 software. For multiple comparisons, one-way ANOVA is followed by Tukey’s test. Data are expressed as mean ± SD. In all cases, significant differences were considered atP < 0.05.


## 3. Results

### 3.1. Lc-pMG36e-vscO Construction

The vscO gene consisting of an open reading frame of 462 bp was obtained from genomic DNA ofV. alginolyticus by PCR amplification and cloned into the expression plasmid pMG36 generating pMG36e-vscO. The recombinant plasmid pMG36e-vscO was transferred into L. lactis by electroporation. The recombinant L. lactis containing plasmid pMG36e-vscO was named Lc-pMG36e-vscO. To determine whether the L. lactis is capable of producing VscO protein, whole-cell lysates of Lc-pMG36e-vscO were analyzed by western blotting after SDS-PAGE. The result showed that an immunoreactive band of about 18 kDa was detected in the recombinant strain (Figure 2), which indicated that the VscO protein was produced by Lc-pMG36e-vscO.Figure 2
Analysis of VscO protein expression inL. lactis. M: protein marker, 1: whole protein of wild L. lactis; 2: whole protein of Lc-pMG36e-vscO.


### 3.2. Hereditary Stability of Lc-pMG36e-vscO

The Lc-pMG36e-vscO gene was screened by PCR and sequence analysis for genetic stability analysis after 50 generations. The results showed that recombinant plasmid pMG36e-vscO was stably inherited in Lc-pMG36e-vscO (Figure3).Figure 3
Hereditary stability analysis of Lc-pMG36e-vscO. M: DL2000 DNA marker; v1–v10: PCR product amplified by primers vsco–R/vsco-F; u1–u10: PCR product amplified by primers pMG36e-R/pMG36e-F. v1-10 and u1-10: 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 generations of Lc-pMG36e-vscO.


### 3.3. Analysis of Serum Antibody Titer

The titers of VscO-specific serum antibody in grouper were shown in Figure4. The results showed that a specific antibody in the immunized grouper was detected at the first week after vaccination. The specific antibody titers in the Lc-pMG36e-vscO group were significantly higher than that in the L. lactis group and PBS group (P < 0.05). The antibody titers displayed an upward tendency from the 1st to the 4th week and then a downward tendency from the 4th week to the end of the experiments. The antibody titers reached the peak value of 9.6 at the 4th week after booster immunization (Figure 4).Figure 4
Antibody titers in sera of grouper immunized with PBS,L. lactis, and Lc-pMG36e-vscO. Sera collected at week 1–7 after vaccination. Each data column represented the mean of log2 (antibody titers) with standard error bar. The asterisks indicated significant differences (∗P < 0.05; ∗∗P < 0.01) between the controls and immunized group.


### 3.4. The Expression Levels of Immune-Related Genes

At 7, 14, 21, 28, 35, and 42 d after oral administration, qRT-PCR was performed to analyze the expression levels of the immune-related gene in the spleen, kidney, and hindgut (Figure5). The results showed IL-1β gene expression levels had increased after the first immunization in the Lc-pMG36e-VscO group and then had declined till the end of experiments in all detected tissues. The expression levels of the TNF-α gene did not change in spleen, increased-declined-increased-declined in the head kidney, and continuously increased in the hindgut during the immunization in the Lc-pMG36e-vscO group. The expression levels of the IL-16 gene in the Lc-pMG36e-vscO group had the trend of increasing-declining-increasing-declining during the immunization in the Lc-pMG36e-vscO group in all detected tissues. The expression levels of the MHC-1α gene displayed the trend of increasing-declining-increasing-declining during the immunization in the Lc-pMG36e-vscO group in spleen and head kidney and had no significant change in hindgut.Figure 5
qRT-PCR analysis of the expression levels of immune-related genes in spleen, head kidney, and intestine of grouper (n = 4 fish/group) after immunization. (a) IL-1β; (b) TNF-α; (c) IL-16; (d) MHC-1α. ∗P < 0.05, ∗∗P < 0.01.
(a)(b)(c)(d)


### 3.5. Challenge Experiments

Challenge experiments were performed by injectingV. alginolyticus virulent strain HY9901 to evaluate the protective effect of Lc-pMG36e-vscO. As shown in Figure 6, the fish immunized with Lc-pMG36e-vscO showed a relative percentage survival (RPS) of 68% after challenge with a lethal dose of V. alginolyticus, significantly higher than those of L. lactis (32.67%) and PBS group (0%).Figure 6
Survival rate of grouper after challenge withV. alginolyticus post immunization. 20 fish/group were used to record percent survival for 15 days. The experiments were replicated in triplicate.


## 3.1. Lc-pMG36e-vscO Construction

The vscO gene consisting of an open reading frame of 462 bp was obtained from genomic DNA ofV. alginolyticus by PCR amplification and cloned into the expression plasmid pMG36 generating pMG36e-vscO. The recombinant plasmid pMG36e-vscO was transferred into L. lactis by electroporation. The recombinant L. lactis containing plasmid pMG36e-vscO was named Lc-pMG36e-vscO. To determine whether the L. lactis is capable of producing VscO protein, whole-cell lysates of Lc-pMG36e-vscO were analyzed by western blotting after SDS-PAGE. The result showed that an immunoreactive band of about 18 kDa was detected in the recombinant strain (Figure 2), which indicated that the VscO protein was produced by Lc-pMG36e-vscO.Figure 2
Analysis of VscO protein expression inL. lactis. M: protein marker, 1: whole protein of wild L. lactis; 2: whole protein of Lc-pMG36e-vscO.


## 3.2. Hereditary Stability of Lc-pMG36e-vscO

The Lc-pMG36e-vscO gene was screened by PCR and sequence analysis for genetic stability analysis after 50 generations. The results showed that recombinant plasmid pMG36e-vscO was stably inherited in Lc-pMG36e-vscO (Figure3).Figure 3
Hereditary stability analysis of Lc-pMG36e-vscO. M: DL2000 DNA marker; v1–v10: PCR product amplified by primers vsco–R/vsco-F; u1–u10: PCR product amplified by primers pMG36e-R/pMG36e-F. v1-10 and u1-10: 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 generations of Lc-pMG36e-vscO.


## 3.3. Analysis of Serum Antibody Titer

The titers of VscO-specific serum antibody in grouper were shown in Figure4. The results showed that a specific antibody in the immunized grouper was detected at the first week after vaccination. The specific antibody titers in the Lc-pMG36e-vscO group were significantly higher than that in the L. lactis group and PBS group (P < 0.05). The antibody titers displayed an upward tendency from the 1st to the 4th week and then a downward tendency from the 4th week to the end of the experiments. The antibody titers reached the peak value of 9.6 at the 4th week after booster immunization (Figure 4).Figure 4
Antibody titers in sera of grouper immunized with PBS,L. lactis, and Lc-pMG36e-vscO. Sera collected at week 1–7 after vaccination. Each data column represented the mean of log2 (antibody titers) with standard error bar. The asterisks indicated significant differences (∗P < 0.05; ∗∗P < 0.01) between the controls and immunized group.


## 3.4. The Expression Levels of Immune-Related Genes

At 7, 14, 21, 28, 35, and 42 d after oral administration, qRT-PCR was performed to analyze the expression levels of the immune-related gene in the spleen, kidney, and hindgut (Figure5). The results showed IL-1β gene expression levels had increased after the first immunization in the Lc-pMG36e-VscO group and then had declined till the end of experiments in all detected tissues. The expression levels of the TNF-α gene did not change in spleen, increased-declined-increased-declined in the head kidney, and continuously increased in the hindgut during the immunization in the Lc-pMG36e-vscO group. The expression levels of the IL-16 gene in the Lc-pMG36e-vscO group had the trend of increasing-declining-increasing-declining during the immunization in the Lc-pMG36e-vscO group in all detected tissues. The expression levels of the MHC-1α gene displayed the trend of increasing-declining-increasing-declining during the immunization in the Lc-pMG36e-vscO group in spleen and head kidney and had no significant change in hindgut.Figure 5
qRT-PCR analysis of the expression levels of immune-related genes in spleen, head kidney, and intestine of grouper (n = 4 fish/group) after immunization. (a) IL-1β; (b) TNF-α; (c) IL-16; (d) MHC-1α. ∗P < 0.05, ∗∗P < 0.01.
(a)(b)(c)(d)


## 3.5. Challenge Experiments

Challenge experiments were performed by injectingV. alginolyticus virulent strain HY9901 to evaluate the protective effect of Lc-pMG36e-vscO. As shown in Figure 6, the fish immunized with Lc-pMG36e-vscO showed a relative percentage survival (RPS) of 68% after challenge with a lethal dose of V. alginolyticus, significantly higher than those of L. lactis (32.67%) and PBS group (0%).Figure 6
Survival rate of grouper after challenge withV. alginolyticus post immunization. 20 fish/group were used to record percent survival for 15 days. The experiments were replicated in triplicate.


## 4. Discussion

Vibrio spp is a part of normal marine flora. However, it also is a pathogen of aquaculture animals and leads to massive mortality of aquaculture animals in hatcheries and grow-out farms [1]. V. alginolyticus is the pathogen of vibriosis of several aquaculture animals [2–7]. Co-infection of V. alginolyticus and other pathogen bacteria also caused disease in fish [25, 26]. L. calcarifer infected with V. alginolyticus moved in circles on the water surface of the cage. Clinical symptoms of affected fish included congestion, haemorrhage, and vacuolation in liver [4]. In this paper, all fish in the control group died after 8 d of being challenged by V. alginolyticus. Vaccination is playing an increasingly important role in the control of fish diseases. Till now, many experimental vaccines against V. alginolyticus have been developed [12, 27, 28]. However, these vaccines have not been used on a large scale because of the stress induced during the vaccination or side effects post vaccination in practical application. Therefore, an oral vaccine with fewer side effects aroused widespread concern [11].Many pathogenic microorganisms initiate their infectious cycle at the mucosal surfaces of fish. Appropriate mucosal stimulation can evoke the effective protection of mucosal surfaces from colonization and invasion by infectious pathogens [29]. Therefore, the vaccine antigens need to be delivered to various mucosal sites [29]. Probiotics as heterologous antigen carriers can confer protective immunity, which is a potential way to develop a mucosal vaccine [18, 24, 30]. In this study, L. lactis was isolated from the intestine of pearl gentian grouper and it can adhere to the mucosal surfaces of the intestine of fish, which is conducive to the delivery of the antigen protein expressed in L. lactis to the mucosal surfaces of the intestine. The antigen molecules used in vaccine development must have high conservation and immunogenicity and can be easily recognized by antigen-presenting cells [31, 32]. Outer membrane proteins (OmpU, OmpK, and OmpW) of V. alginolyticus are very antigenic and can be used as vaccine candidates [21, 32]. It is sufficiently evident that the VscO protein of V. alginolyticushas good immunogenicity and may be used as a candidate antigen for developing a vaccine [12]. In this study, a recombinant L. lactis expressing V. alginolyticus VscO protein is constructed. The recombinant plasmid was stably inherited, and the VscO protein was stably expressed on L. lactis. The result is consistent with other reports [16, 18, 19].Oral immunization with a recombinant vaccine could induce a specific immune response at the mucosal and systemic levels [18, 20]. In the present study, the immunogenicity of Lc-pMG36e-VscO was evaluated by inoculation of grouper via oral immunization. The results showed that Lc-pMG36e-VscO strain has the capacity to induce a higher serum antibody titer when compared with the control L. lactis strain and PBS group. A comparison of the means of log2 (antibody titer) in both the control L. lactis and PBS group revealed that they were not significantly different (P > 0.05). Fish immunized with the rOmpC-vaccinated produced specific anti-rOmpC antibodies with a significant antibody titer [19]. The antibody level of the immunized fish with the recombinant vaccine was significantly higher than that in the control group [18, 20]. Higher antibody titers suggested that antigen induced the release of specific IgM and activated the adaptive immunity of fish.Humoral and mucosal immune responses were demonstrated to eliminate pathogens in fish [33]. Cytokines also play a key role in the regulation of the inflammatory process and serve as an important component of innate immunity. In the present study, the expression of immune-related genes (IL-1β, IL-16, TNF-α, and MHC-1α) in the spleen, kidney, and gut of fish immunized with the Lc-pMG36e-vscO were upregulated compared with the control group. This indicates that oral immunization with Lc-pMG36e-vscO induced a higher immune response in fish. Immunized fish (Carassius auratus) with recombinant L. casei expressing AcrV protein of A. veronii upregulated the expression of IL-1β, TNF-α, IFN-γ, and IL-10 [34]. The expression of IL-10, IL-β, IFN-γ, and TNF-α genes in Cyprinus carpio immunized with recombinant L. casei was significantly upregulated [16]. The expression of part of immune-related genes in the main tissues of fish fed with L. lactis was also up-regulated. L. casei, as a probiotic, can stimulate the innate immune response in fish by increasing the expression of IL-1β and IFN-γ [34]. Live L. lactis can significantly enhance the levels of TNF-α, IL-1β, and IL-6 in C. carpio [15]. The cellular components of probiotics could stimulate immune-related gene expression and may be useful as adjuvants for vaccines in aquaculture [35]. These results indicated that recombinant vaccines can trigger an inflammatory response and cellular immune response, and probiotics also play an immunomodulatory role.In the challenge experiment, the data showed that the survival rates in the Lc-pMG36e-VscO group were significantly higher than those in theL. lactis group and PBS group (P < 0.05). This result was consistent with similar studies involving recombinant OmpAI and flagellin A protein, and OmpC of A. veronii obtained high survival rates [16, 18, 19]. We hypothesized that Lc-pMG36e-vscO can arrive at the mucosal surfaces of the gut and produce antibodies or other inhibitory substances as a barrier against V. alginolyticus.


## 5. Conclusions

In conclusion, the Lc-pMG36e-vscO expressing VscO protein has good immunogenicity. Oral immunization with Lc-pMG36e-VscO in grouper stimulated high serum specific antibody titers and elicited immune response and conferred efficient protection againstV. alginolyticus. These results indicated that recombinant L. lactis may be promising as a candidate vaccine against.V. alginolyticus.



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*Source: 7226451-2023-02-07.xml*