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array:22 [ "pii" => "S0301054617300769" "issn" => "03010546" "doi" => "10.1016/j.aller.2017.04.005" "estado" => "S300" "fechaPublicacion" => "2017-09-01" "aid" => "879" "copyrightAnyo" => "2017" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Allergol Immunopathol (Madr). 2017;45:496-505" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 5 "formatos" => array:2 [ "HTML" => 4 "PDF" => 1 ] ] "itemSiguiente" => array:18 [ "pii" => "S0301054616301653" "issn" => "03010546" "doi" => "10.1016/j.aller.2016.10.012" "estado" => "S300" "fechaPublicacion" => "2017-09-01" "aid" => "827" "copyright" => "SEICAP" "documento" => "article" "crossmark" => 1 "subdocumento" => "sco" "cita" => "Allergol Immunopathol (Madr). 2017;45:506-7" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 21 "formatos" => array:2 [ "HTML" => 17 "PDF" => 4 ] ] "en" => array:11 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Point of view</span>" "titulo" => "Use of anti-allergic drugs in children" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "506" "paginaFinal" => "507" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "C. Suárez-Castañón, G. Modroño-Riaño, G. Solís-Sánchez" "autores" => array:3 [ 0 => array:2 [ "nombre" => "C." "apellidos" => "Suárez-Castañón" ] 1 => array:2 [ "nombre" => "G." "apellidos" => "Modroño-Riaño" ] 2 => array:2 [ "nombre" => "G." "apellidos" => "Solís-Sánchez" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0301054616301653?idApp=UINPBA00004N" "url" => "/03010546/0000004500000005/v1_201709090108/S0301054616301653/v1_201709090108/en/main.assets" ] "itemAnterior" => array:18 [ "pii" => "S0301054617300708" "issn" => "03010546" "doi" => "10.1016/j.aller.2017.02.010" "estado" => "S300" "fechaPublicacion" => "2017-09-01" "aid" => "873" "copyright" => "SEICAP" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Allergol Immunopathol (Madr). 2017;45:487-95" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:2 [ "total" => 5 "formatos" => array:2 [ "HTML" => 4 "PDF" => 1 ] ] "en" => array:12 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original Article</span>" "titulo" => "Trends in prevalence and risk factors of allergic rhinitis symptoms in primary schoolchildren six years apart in Budapest" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "487" "paginaFinal" => "495" ] ] "contieneResumen" => array:1 [ "en" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1184 "Ancho" => 2703 "Tamanyo" => 139064 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Feather bedding and family history of atopy. AR+: children with allergic rhinitis symptoms; AR−: children without allergic rhinitis symptoms; family history of atopy+: children with family history of atopy; family history of atopy−: children without family history of atopy; <span class="elsevierStyleItalic">n</span>: number of children.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "M. Sultész, I. Balogh, G. Katona, G. Mezei, A. Hirschberg, G. Gálffy" "autores" => array:6 [ 0 => array:2 [ "nombre" => "M." "apellidos" => "Sultész" ] 1 => array:2 [ "nombre" => "I." "apellidos" => "Balogh" ] 2 => array:2 [ "nombre" => "G." "apellidos" => "Katona" ] 3 => array:2 [ "nombre" => "G." "apellidos" => "Mezei" ] 4 => array:2 [ "nombre" => "A." "apellidos" => "Hirschberg" ] 5 => array:2 [ "nombre" => "G." "apellidos" => "Gálffy" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0301054617300708?idApp=UINPBA00004N" "url" => "/03010546/0000004500000005/v1_201709090108/S0301054617300708/v1_201709090108/en/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original Article</span>" "titulo" => "Oral immunisation with Taishan <span class="elsevierStyleItalic">Pinus massoniana</span> pollen polysaccharide adjuvant with recombinant <span class="elsevierStyleItalic">Lactococcus lactis</span>-expressing <span class="elsevierStyleItalic">Proteus mirabilis</span> ompA confers optimal protection in mice" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "496" "paginaFinal" => "505" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "J. Zhou, K. Wei, C. Wang, W. Dong, N. Ma, L. Zhu, L.P. Hu, H. Huang, R. Zhu" "autores" => array:9 [ 0 => array:3 [ "nombre" => "J." "apellidos" => "Zhou" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "K." "apellidos" => "Wei" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 2 => array:3 [ "nombre" => "C." "apellidos" => "Wang" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 3 => array:3 [ "nombre" => "W." "apellidos" => "Dong" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 4 => array:3 [ "nombre" => "N." "apellidos" => "Ma" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 5 => array:3 [ "nombre" => "L." "apellidos" => "Zhu" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 6 => array:3 [ "nombre" => "L.P." "apellidos" => "Hu" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 7 => array:3 [ "nombre" => "H." "apellidos" => "Huang" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">c</span>" "identificador" => "aff0015" ] ] ] 8 => array:4 [ "nombre" => "R." "apellidos" => "Zhu" "email" => array:1 [ 0 => "zhurl@sdau.edu.cn" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Technology, Shandong Agricultural University, Shandong Taian 271018, PR China" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Animal Disease Prevention and Control Center of Shandong Province, Animal Husbandry and Veterinary Bureau of Shandong Province, Shandong Jinan 250022, PR China" "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:3 [ "entidad" => "Shandong New Hope Liuhe Co., Ltd, New Hope Group, Shandong Qingdao, 266061, PR China" "etiqueta" => "c" "identificador" => "aff0015" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 900 "Ancho" => 3339 "Tamanyo" => 106327 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Flow cytometric and immunofluorescence analysis of the <span class="elsevierStyleItalic">L. lactis</span> expressing ompA. (A) Flow cytometric analysis of the <span class="elsevierStyleItalic">L. lactis</span> expressing ompA. The mouse anti-ompA monoclonal antibody was used in this assay. The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> showed a significant increase of fluorescence intensity (green); the blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> showed negative fluorescence (red). (B and C) Immunofluorescence analysis of the <span class="elsevierStyleItalic">L. lactis</span> expressing ompA. The mouse anti-ompA monoclonal antibody was used in this assay. The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> showed positive green fluorescence on the cells (B). No fluorescence was shown in the blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> (C).</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Proteus mirabilis</span> is a well-known zoonotic pathogen that widely exists in nature<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">1</span></a>; this pathogen occurs at an unprecedented rate in animal and human populations and is a major cause of consternation for public health and veterinary communities. <span class="elsevierStyleItalic">P. mirabilis</span> can infect various animals and induce clinical symptoms, such as diarrhoea, sepsis, muscle erosion, and encephalomalacia. The pathogen is also responsible for skin ulcers and causes high mortality and economic losses in aquatic animals.<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">2</span></a> In humans, <span class="elsevierStyleItalic">P. mirabilis</span> can cause ascending opportunistic and nosocomial urinary tract infections, which occur in patients with functional or structural abnormalities in the urinary tract.<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">3</span></a> In animal husbandry, <span class="elsevierStyleItalic">P. mirabilis</span> leads to serious diarrhoea and death of lambs as well as miscarriage of pregnant sheep in large-scale sheep farms.<a class="elsevierStyleCrossRefs" href="#bib0170"><span class="elsevierStyleSup">4,5</span></a> The use of antibiotics in controlling <span class="elsevierStyleItalic">P. mirabilis</span> infection has not been recommended because of health hazards to consumers and induced multidrug resistance of pathogenic bacteria. However, few commercial vaccines for animals are available currently. Given the high morbidity rates associated with <span class="elsevierStyleItalic">P. mirabilis</span> infections and the limited therapeutic options, scholars have focused on developing a safe and effective vaccine against <span class="elsevierStyleItalic">P. mirabilis</span> for the breeding industry.</p><p id="par0010" class="elsevierStylePara elsevierViewall">Mucosa tissues are important for protection of an organism from diseases caused by viral, bacterial, and parasitic pathogens, which invade the body through the mucosal system.<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">6</span></a> However, vaccines administered by parenteral routes generally fail to induce mucosal immune responses. Therefore, oral vaccination can be an efficient approach for interfering the colonisation of enteropathogenic bacteria; this strategy can effectively induce local immune responses at the intestinal mucosa and concurrently elicit systemic immune responses.<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">7</span></a> Nevertheless, orally administered antigens must survive the harsh acidic environment and attack of proteases to interact with the immune tissues of the gut and induce immune responses.<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">6</span></a><span class="elsevierStyleItalic">Lactic acid bacteria</span> (LAB) are traditionally used in food industry and generally regarded as safe for human consumption. <span class="elsevierStyleItalic">Lactococcus lactis</span> is a model LAB that has been extensively studied for oral vaccine delivery. <span class="elsevierStyleItalic">L. lactis</span> is used to express some bacterial, viral, and parasitic antigens, and the resultant recombinant strains can induce specific mucosal and systemic immune responses upon oral administration.<a class="elsevierStyleCrossRefs" href="#bib0190"><span class="elsevierStyleSup">8–10</span></a> In <span class="elsevierStyleItalic">L. lactis</span> expression mode, a Nisin-controlled gene expression system (NICE) can transport the foreign protein to the bacterial cell surface; this mode is an effective and multifunctional tool.<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">11</span></a> Thus, oral immunisation with <span class="elsevierStyleItalic">L. lactis</span> carriers exhibits potential for prevention of <span class="elsevierStyleItalic">P. mirabilis</span> infection.</p><p id="par0015" class="elsevierStylePara elsevierViewall">In this study, we constructed a recombinant <span class="elsevierStyleItalic">L. lactis</span> expressing <span class="elsevierStyleItalic">P. mirabilis</span> outer membrane protein A (ompA), one of the main protective antigens in <span class="elsevierStyleItalic">P. mirabilis</span>.<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> We also evaluated the specific protection conferred by the recombinant strain against bacterial challenge in mice. Administration of a mucosal adjuvant can augment both mucosal and systemic immune responses to vaccine antigens.</p><p id="par0020" class="elsevierStylePara elsevierViewall">Taishan <span class="elsevierStyleItalic">Pinus massoniana</span> pollen polysaccharides (TPPPS), a pleiotropic polysaccharide extracted from Taishan <span class="elsevierStyleItalic">P. massoniana</span> pollens, has been studied in our laboratory since 2003; TPPPS can be used as an effective adjuvant to improve the immune system and facilitate immune responses.<a class="elsevierStyleCrossRefs" href="#bib0215"><span class="elsevierStyleSup">13,14</span></a> In the present study, TPPPS was first used as an oral vaccine adjuvant. The effects of TPPPS on conditioning intestine mucosal immunity were also investigated.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Methods and materials</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Ethics statement</span><p id="par0025" class="elsevierStylePara elsevierViewall">The animal procedures used in this study were approved by the Animal Care and Use Committee of Shandong Agricultural University (permit number: 20010510) and performed in accordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China).</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Bacterial strain, vector, and medium</span><p id="par0030" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">P. mirabilis</span> strain PM.1 was isolated from a dead lamb with diarrhoea in 2013 (Shandong, China) and then preserved in our laboratory. The plasmid pNZ8149 and <span class="elsevierStyleItalic">L. lactis</span> NZ3900 strain were purchased from MoBiTec GmbH (Goettingen, Germany). <span class="elsevierStyleItalic">Escherichia coli</span> DH5α and pMD18-T were purchased from Takara Co., Ltd., China. Elliker-medium [yeast extract, 5<span class="elsevierStyleHsp" style=""></span>g/L; tryptone, 20<span class="elsevierStyleHsp" style=""></span>g/L; NaCl, 4<span class="elsevierStyleHsp" style=""></span>g/L; CH<span class="elsevierStyleInf">3</span>COONa, 1.5<span class="elsevierStyleHsp" style=""></span>g/L; L(+) ascorbic acid, 0.5<span class="elsevierStyleHsp" style=""></span>g/L; agar, 15<span class="elsevierStyleHsp" style=""></span>g/L; 2 and bromocresol purple 0.5%, pH<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>6.8] and M17 broth medium containing 0.5% lactose were purchased from Sigma (Beijing, China). All yeast culture media were prepared in accordance with the manufacturers’ guidelines.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Construction of recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span></span><p id="par0035" class="elsevierStylePara elsevierViewall">On the basis of the ompA gene sequence of <span class="elsevierStyleItalic">P. mirabilis</span> (GenBank Type: RefSeq (Nucleotide) <a href="ncbi-n:NC_010554.1">NC_010554.1</a>), a pair of primers (ompA-F: 5′-C<span class="elsevierStyleUnderline">CCATGG</span>G TATGATAACGAGGCGTAAAATGAAAAAGACAGCTATCGCATTAGCAG-3′, ompA-R: 5′-CTGC<span class="elsevierStyleUnderline">TCTAGA</span>TTAGTGACCAGGTTGAACAACAAC-3′) was designed to produce a 1107<span class="elsevierStyleHsp" style=""></span>bp fragment by polymerase chain reaction (PCR). The PCR product was then digested with the restriction enzymes Nco I and Xba I, and the digested fragment was cloned into plasmid pNZ8149. The resultant plasmid was confirmed by sequencing (Sunny, Shanghai) and transformed into <span class="elsevierStyleItalic">L. lactis</span> NZ3900 (named pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>). The blank pNZ8149 plasmid was transformed into <span class="elsevierStyleItalic">L. lactis</span> NZ3900 as negative control and named blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span>.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Protein expression, flow cytometry, and immunofluorescence microscopy</span><p id="par0040" class="elsevierStylePara elsevierViewall">Recombinant <span class="elsevierStyleItalic">L. lactis</span> NZ3900 cells were cultured in M17 broth medium containing 0.5% lactose as carbon source at 30<span class="elsevierStyleHsp" style=""></span>°C overnight under anaerobic conditions. Recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> cells were diluted by 1:25 to 50 in M17 medium. When the cell density reached 0.4 of OD<span class="elsevierStyleInf">600</span>, Nisin was added every six hours to ensure continuous induction up to a final concentration of 10<span class="elsevierStyleHsp" style=""></span>ng/mL. Western blot analyses were performed to identify ompA by using methods described in previous study.<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> The mouse anti-ompA protein monoclonal antibody used in Western blot analysis was prepared in the laboratory.</p><p id="par0045" class="elsevierStylePara elsevierViewall">The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> and blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> were centrifuged at 5000<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min at 4<span class="elsevierStyleHsp" style=""></span>°C and washed thrice with sterile phosphate-buffered saline (PBS) to investigate whether the ompA protein was expressed on the surface of <span class="elsevierStyleItalic">L. lactis</span>. The bacteria were then incubated with mouse anti-ompA protein monoclonal antibody at 4<span class="elsevierStyleHsp" style=""></span>°C overnight, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma, China). The stained cells were analysed by flow cytometry (Guaga Easy Cyte Mini, USA).</p><p id="par0050" class="elsevierStylePara elsevierViewall">For immunofluorescence staining, the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> and blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> were harvested after induction and incubated with mouse anti-ompA monoclonal antibody as the primary antibody followed by FITC-conjugated goat anti-mouse IgG (Sigma, China) as the secondary antibody. The blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> was used as negative control.</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Vaccine preparation</span><p id="par0055" class="elsevierStylePara elsevierViewall">The cultured recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> was resuspended in sterile PBS at a concentration of 1<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">11</span> colony-forming unit (CFU)/mL (100<span class="elsevierStyleHsp" style=""></span>μL/mouse). The corresponding blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> was treated similarly.</p><p id="par0060" class="elsevierStylePara elsevierViewall">TPPPS was prepared in our laboratory through water extraction and ethanol precipitation.<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">13</span></a> The contents of TPPPS were set at the following three doses: 50 (low), 100 (moderate), and 200 (high) mg/mL in three separate TPPPS adjuvant vaccines. The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> mixed with three doses of TPPPS was separately prepared to obtain the corresponding adjuvant oral vaccine.</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Animal experiment</span><p id="par0065" class="elsevierStylePara elsevierViewall">A total of 360 six-week-old SPF BALB/c mice (female; Spirax Ferrer Poultry Co., Ltd, Jinan) were randomly separated into six sterilised isolators (groups I–VI), with 60 mice each. The ambient conditions were set to 20–25<span class="elsevierStyleHsp" style=""></span>°C and 30–40% relative humidity. Air entering the isolators was filtered. Mice in groups I–VI were inoculated orally with 0.1<span class="elsevierStyleHsp" style=""></span>mL of low, moderate, and high doses of TPPPS adjuvant recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, pure pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span>, and PBS. Groups I–VI were named pNZ-ompA-TPPPS (L), pNZ-ompA-TPPPS (M), pNZ-ompA-TPPPS (H), pNZ-ompA, blank-pNZ, and Mock, respectively. The vaccines were inoculated daily at 0–4<span class="elsevierStyleHsp" style=""></span>dpi (days post the first inoculation). Two booster immunisations were conducted at 10–14<span class="elsevierStyleHsp" style=""></span>dpi and 24–28<span class="elsevierStyleHsp" style=""></span>dpi.</p><p id="par0070" class="elsevierStylePara elsevierViewall">At 0, 14, 28, 42, and 56<span class="elsevierStyleHsp" style=""></span>dpi, three mice in each group were selected randomly to determine the antibody titres and the concentrations of IL-2, IFN-γ, IL-4, and interleukin 10 (IL-10) in serum, as well as T-cell proliferative response (LTRs) and counts of CD4+ and CD8+ T lymphocytes in peripheral blood. The tracheal and intestinal lavage fluids were collected to determine sIgA titres by using methods described in previous study.<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">15</span></a> The animals were starved for 12<span class="elsevierStyleHsp" style=""></span>h before sampling.</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Detection of specific IgG and sIgA antibodies as well as IL-2, IFN-γ, IL-4, and IL-10</span><p id="par0075" class="elsevierStylePara elsevierViewall">Three sera, tracheal, and intestinal lavage fluid samples were randomly collected from each group during sampling. Standard enzyme-linked immunosorbent assay (ELISA) protocol was performed for specific antibody IgG titres in serum and sIgA titres in intestinal and tracheal lavage samples.<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">16</span></a> The concentrations of IL-2, IFN-γ, IL-4, and IL-10 were detected using mouse IL-2, IFN-γ, IL-4, and IL-10 ELISA kits (Langdon Bio-technology Co., Ltd, Shanghai) in accordance with the manufacturer's instructions. Absorbance was determined with a microplate reader at 450<span class="elsevierStyleHsp" style=""></span>nm.</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Counts of CD4+ and CD8+ T lymphocytes in peripheral blood</span><p id="par0080" class="elsevierStylePara elsevierViewall">The fresh anticoagulant-heart blood was collected, and then the lymphocytes were obtained with lymphocyte separation medium (P8620-200, Solarbio, China) after centrifugation at 2000<span class="elsevierStyleHsp" style=""></span>rpm for 10<span class="elsevierStyleHsp" style=""></span>min. Then, 10<span class="elsevierStyleHsp" style=""></span>μL of fluorescein isothiocyanate anti-mouse CD4 antibody (BioLegend, USA) and 10<span class="elsevierStyleHsp" style=""></span>μL of phycoerythrin anti-mouse CD8 antibody (BioLegend, USA) were decanted into 50<span class="elsevierStyleHsp" style=""></span>μL of lymphocyte suspension. The mixture was incubated for 20<span class="elsevierStyleHsp" style=""></span>min at 4<span class="elsevierStyleHsp" style=""></span>°C.<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">17</span></a> The percentages of CD4+ and CD8+ T lymphocytes were detected through flow cytometry (Guaga Easy Cyte Mini, USA) in accordance with the manufacturer's instructions.</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Peripheral blood lymphocyte proliferation</span><p id="par0085" class="elsevierStylePara elsevierViewall">Fresh anti-coagulated peripheral blood samples were collected from the mice selected in each group and used to separate lymphocytes as previously described.<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">18</span></a></p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Challenge with <span class="elsevierStyleItalic">P. mirabilis</span></span><p id="par0090" class="elsevierStylePara elsevierViewall">At one day after the last immunisation, 20 mice from each group were challenged orally with LD<span class="elsevierStyleInf">50</span> of <span class="elsevierStyleItalic">P. mirabilis</span>. The number of <span class="elsevierStyleItalic">P. mirabilis</span> colonies in the intestine was determined. Faecal excretion of <span class="elsevierStyleItalic">P. mirabilis</span> was monitored every two days, and serial dilutions of the samples were plated in blood agar. α-Haemolytic colonies were determined after incubation of the plates for 24<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>°C.</p><p id="par0095" class="elsevierStylePara elsevierViewall">Twenty mice from each group were challenged with 10 LD<span class="elsevierStyleInf">50</span><span class="elsevierStyleItalic">P. mirabilis</span> two weeks after the last immunisation. Mice were maintained for seven days post challenge, and deaths were recorded every day. The survival status of mice was calculated with the following formula:Survival rate (%)<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>No. of surviving mice/Total No.<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>100</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Statistical analysis</span><p id="par0100" class="elsevierStylePara elsevierViewall">Data were presented as mean<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>standard deviation (SD), and Duncan's multiple-range test was performed to analyse differences among groups by using SPSS 17.0 software. A <span class="elsevierStyleItalic">P</span>-value of<span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05 was considered statistically significant.</p></span></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Results</span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Construction of recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> and ompA protein expression in vitro</span><p id="par0105" class="elsevierStylePara elsevierViewall">The recombinant plasmid pNZ-ompA was first constructed. The insertion of the ompA gene into the pNZ8149 plasmid was confirmed by restriction enzyme digestion (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>A). Analysis of the phenotypic screening of recombinant <span class="elsevierStyleItalic">L. lactis</span> utilising lactose in Elliker medium showed that the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> presented yellow colonies (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>B). The molecular size of the target protein was approximately 45<span class="elsevierStyleHsp" style=""></span>kDa. Western blot analysis indicated the lack of the clear band in blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> used as control (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>C).</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0110" class="elsevierStylePara elsevierViewall">To determine whether the ompA protein was expressed on the surface of <span class="elsevierStyleItalic">L. lactis</span>, we performed flow cytometric analysis. The results showed that the fluorescence intensity significantly increased in the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A). Immunofluorescence analysis further showed that the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> was immunostained positive for ompA (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>B), but blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> did not (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>C). Hence, the constructed recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> could effectively display the ompA protein on the <span class="elsevierStyleItalic">L. lactis</span> surface.</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">TPPPS promoted mucosal and humoral immune responses induced by oral immunisation of the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span></span><p id="par0115" class="elsevierStylePara elsevierViewall">Mucosal antibodies play an important role in protection against pathogens. In this study, sIgA responses in intestinal and tracheal lavage fluid samples were analysed by ELISA. The anti-ompA sIgA levels in the pNZ-ompA group are significantly higher than those in the control groups inoculated with blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> or PBS in the intestine and trachea at 28–56<span class="elsevierStyleHsp" style=""></span>dpi (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>A and B; <span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). Notably, a significantly higher anti-ompA sIgA response was detected in groups pNZ-ompA-TPPPS (L), (M), and (H) than that in group pNZ-ompA. The group pNZ-ompA-TPPPS (H) produced the highest anti-ompA sIgA titres in the intestine and trachea at 28–42<span class="elsevierStyleHsp" style=""></span>dpi (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05).</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0120" class="elsevierStylePara elsevierViewall">Anti-ompA IgG responses were evaluated in serum samples. The results showed that the anti-ompA IgG titres in mice vaccinated with pNZ-ompA are significantly higher than those in the control groups inoculated with blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> or PBS at 28–56<span class="elsevierStyleHsp" style=""></span>dpi (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>C; <span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). Mice in groups pNZ-ompA-TPPPS (L), (M), and (H) exhibited significantly higher levels of anti-ompA IgG than those that received pure pNZ-ompA at 28–42<span class="elsevierStyleHsp" style=""></span>dpi compared with the TPPPS adjuvant formulations (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). Group pNZ-ompA-TPPPS (H) showed increased IgG production compared with pNZ-ompA-TPPPS (L) and (M) at 28–56<span class="elsevierStyleHsp" style=""></span>dpi (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05).</p><p id="par0125" class="elsevierStylePara elsevierViewall">These results showed that the constructed recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> can elicit both ompA-specific mucosal and systemic antibody responses via oral inoculation. Additionally, TPPPS promoted antibody titres induced by pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> in mice, especially at a concentration of 200<span class="elsevierStyleHsp" style=""></span>mg/mL.</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">TPPPS promoted cell-mediated immune responses to the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span></span><p id="par0130" class="elsevierStylePara elsevierViewall">Cytokines IL-2, INF-γ, IL-4, and IL-10 in serum were determined to characterise cellular immune responses induced by oral immunisation with the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>. Immunised mice in group pNZ-ompA produced significantly higher IL-2, INF-γ, and IL-4 levels than those inoculated with blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> or PBS at 28–56<span class="elsevierStyleHsp" style=""></span>dpi (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>A–C; <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05). Moreover, IL-2, INF-γ, and IL-4 secretion was significantly enhanced in groups pNZ-ompA-TPPPS (L), (M), and (H) compared with that in group pNZ-ompA without TPPPS (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). Notably, group pNZ-ompA-TPPPS (H) showed increased IL-2, INF-γ, and IL-4 levels compared with groups pNZ-ompA-TPPPS (L) and (M) (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). However, IL-10 production in the six groups showed no significant differences (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>D; <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>><span class="elsevierStyleHsp" style=""></span>0.05).</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><p id="par0135" class="elsevierStylePara elsevierViewall">The ratio of lymphocyte proliferation is commonly used to evaluate cellular immunity.<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">19</span></a> In the present study, we found that mice in group pNZ-ompA showed significantly higher LTRs than control mice that received blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> or PBS at 14–56<span class="elsevierStyleHsp" style=""></span>dpi (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>A; <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05). By contrast, the LTRs in mice immunised with TPPPS adjuvant pNZ-ompA are higher than those immunised with pNZ-ompA alone at 28–56<span class="elsevierStyleHsp" style=""></span>dpi (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). The LTRs were the highest in mice immunised with pNZ-ompA-TPPPS (H) but were not significantly different from those in groups pNZ-ompA-TPPPS (L) and (M) (<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>><span class="elsevierStyleHsp" style=""></span>0.05). The number of CD4+ and CD8+ T lymphocytes in serum directly reflects immune function in animals.<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> Overall, the counts of T lymphocytes showed similar trends to that of LTRs (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>B and C). In these two detection indices, the optimal effects were obtained when at 200<span class="elsevierStyleHsp" style=""></span>mg/mL TPPPS was used.</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Inhibition of <span class="elsevierStyleItalic">P. mirabilis</span> colonisation and infection of BALB/c mice after vaccination with the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span></span><p id="par0140" class="elsevierStylePara elsevierViewall">To evaluate the resistance of pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> on <span class="elsevierStyleItalic">P. mirabilis</span> colonisation, we challenged the mice with <span class="elsevierStyleItalic">P. mirabilis</span> 1 d after the last immunisation. <span class="elsevierStyleItalic">P. mirabilis</span> colonisation was significantly reduced in the small intestine in group pNZ-ompA compared with those in groups blank-pNZ and Mock (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>A; <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05). Interestingly, the administration of TPPPS adjuvant pNZ-ompA conferred improved inhibition on <span class="elsevierStyleItalic">P. mirabilis</span> colonisation than pNZ-ompA administration alone (<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05). Group pNZ-ompA-TPPPS (H) showed the highest resistance to <span class="elsevierStyleItalic">P. mirabilis</span> colonisation (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05).</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia><p id="par0145" class="elsevierStylePara elsevierViewall">Furthermore, we examined the protection of the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> on mice challenged with <span class="elsevierStyleItalic">P. mirabilis</span>. As shown in <a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>B, the protection rate in mice reached 100% in group pNZ-ompA-TPPPS (H), 85% in group pNZ-ompA-TPPPS (M), 75% in group pNZ-ompA-TPPPS (L), and 70% in pure pNZ-ompA (<span class="elsevierStyleItalic">P<span class="elsevierStyleHsp" style=""></span><</span><span class="elsevierStyleHsp" style=""></span>0.05). No mouse survived in groups blank-pNZ and Mock at four days post-challenge. Hence, the pure recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> protects mice against <span class="elsevierStyleItalic">P. mirabilis</span> challenge limitedly. However, addition of 200<span class="elsevierStyleHsp" style=""></span>mg/mL TPPPS to pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> can completely protect mice against <span class="elsevierStyleItalic">P. mirabilis</span> infection.</p></span></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Discussion</span><p id="par0150" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">P. mirabilis</span> is an enteric pathogenic bacteria that frequently causes animal infections and is thus considered as pathogenic diarrheagenic bacteria.<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">21</span></a> Protective immunity against <span class="elsevierStyleItalic">P. mirabilis</span> mainly depends on specific mucosal immune responses induced by intestinal submucosal lymphoid tissues.<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">22</span></a> For mucosal vaccination, LAB are widely used as live vaccine vehicles against various microbes because they present heterologous epitopes, thereby facilitating recognition by the immune system and mediating an immunoadjuvant effect with some of its components.<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">23</span></a> In the present study, we constructed <span class="elsevierStyleItalic">L. lactis</span> harbouring the recombinant pNZ8149-ompA plasmid, which expressed exogenous <span class="elsevierStyleItalic">P. mirabilis</span> ompA protein based on a Nisin-controlled gene expression system. We also evaluated the immunogenicity of this recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> in mice. TPPPS was first used as adjuvant to examine its immune enhancement effects on the oral vaccine. Our results demonstrated that oral immunisation with the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> can elicit both mucosal sIgA and systemic IgG immune responses. Furthermore, TPPPS adjuvant increased immunity levels induced by the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>.</p><p id="par0155" class="elsevierStylePara elsevierViewall">Several expression systems are available for regulated and constitutive expression in <span class="elsevierStyleItalic">L. lactis</span><a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">24</span></a>; NICE system is the most often used. Nisin-regulated gene expression in <span class="elsevierStyleItalic">L. lactis</span> exhibits numerous characteristics: (1) overexpression of homologous and heterologous genes for functional studies to obtain large quantities of specific gene products; (2) metabolic engineering; (3) expression of prokaryotic and eukaryotic membrane proteins; and (4) protein secretion and anchoring in the cell envelope.<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">11</span></a> This expression system is highly versatile and exhibits potential in pharmaceutical, medical, and food technology fields.<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">25</span></a> In the past 10 years, some immune functional proteins expressed by the NICE system in <span class="elsevierStyleItalic">L. lactis</span> can be transported to the bacterial cell surface; consequently, both mucosal and systemic immune responses in the body conferred protection against pathogens.<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">26</span></a></p><p id="par0160" class="elsevierStylePara elsevierViewall">The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> can also significantly increase the production of IL-2, IFN-γ, and IL-4 in mice. IL-2, and IFN-γ, which belong to the Th1 cell cytokine family, play an important role in mediating cytotoxic effects and local inflammatory responses, assisting antibody generation, and participating in cellular immune responses.<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">27</span></a> IL-4, as a Th2 cell cytokine, mainly promotes B cell proliferation and mediates humoral immune responses. However, we observed no significant differences on secretion of IL-10, which inhibits pro-inflammatory cytokine production and prevents macrophage apoptosis and tissue damage.<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">28</span></a> This result demonstrated that a mixed Th1/Th2-based cell-mediated immune response was upregulated, and IL-10-mediated immunosuppression can be avoided through the common mucosal immune system during oral mouse immunisation with the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>.</p><p id="par0165" class="elsevierStylePara elsevierViewall">Adjuvants are widely applied to enhance the immunogenicity of oral vaccines.<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">29</span></a> Taishan <span class="elsevierStyleItalic">P. massoniana</span> pollens have been used as traditional medicine for thousands of years in China and are considered effective adjuvants for improving the immune system and facilitating immune responses in our laboratory.<a class="elsevierStyleCrossRefs" href="#bib0215"><span class="elsevierStyleSup">13,14</span></a> TPPPS contains three kinds of polysaccharides (named TPPPS1–3), and each component is composed of different monosaccharides and shows different dominant activities in anti-oxidant, anti-virus, and immunomodulation; hence, TPPPS exhibit synergistic effects on facilitating the immune function of organisms.<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">30</span></a> As such, the use of TPPPS as adjuvant presented satisfactory effects on improving the immune responses of the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> oral vaccine by enhancing both mucosal and systemic immunity.</p><p id="par0170" class="elsevierStylePara elsevierViewall">In conclusion, we demonstrated that orally-administered recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> survives the transit of the upper gastrointestinal tract as well as expresses and secretes heterologous proteins, which induced specific mucosal and system immune responses against <span class="elsevierStyleItalic">P. mirabilis</span>. Moreover, TPPPS adjuvant presented good immune-enhancing effects on orally administered pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>. This study presents the potential of the recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> combined with TPPPS adjuvant on preventing <span class="elsevierStyleItalic">P. mirabilis</span> infection.</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Ethical disclosures</span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Confidentiality of data</span><p id="par0175" class="elsevierStylePara elsevierViewall">The authors declare that they have followed the protocols of their work centre on the publication of patient data and that all the patients included in the study have received sufficient information and have given their informed consent in writing to participate in that study.</p></span><span id="sec0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Right to privacy and informed consent</span><p id="par0180" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article.</p></span><span id="sec0115" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Protection of human subjects and animals in research</span><p id="par0185" class="elsevierStylePara elsevierViewall">The authors declare that the procedures followed were in accordance with the regulations of the responsible Clinical Research Ethics Committee and in accordance with those of the World Medical Association and the Helsinki Declaration.</p></span></span><span id="sec0120" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Conflict of interest</span><p id="par0190" class="elsevierStylePara elsevierViewall">None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres898652" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Method" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec879393" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 3 => array:3 [ "identificador" => "sec0010" "titulo" => "Methods and materials" "secciones" => array:11 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Ethics statement" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Bacterial strain, vector, and medium" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Construction of recombinant pNZ-ompA/L. lactis" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Protein expression, flow cytometry, and immunofluorescence microscopy" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Vaccine preparation" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Animal experiment" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Detection of specific IgG and sIgA antibodies as well as IL-2, IFN-γ, IL-4, and IL-10" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Counts of CD4+ and CD8+ T lymphocytes in peripheral blood" ] 8 => array:2 [ "identificador" => "sec0055" "titulo" => "Peripheral blood lymphocyte proliferation" ] 9 => array:2 [ "identificador" => "sec0060" "titulo" => "Challenge with P. mirabilis" ] 10 => array:2 [ "identificador" => "sec0065" "titulo" => "Statistical analysis" ] ] ] 4 => array:3 [ "identificador" => "sec0070" "titulo" => "Results" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0075" "titulo" => "Construction of recombinant pNZ-ompA/L. lactis and ompA protein expression in vitro" ] 1 => array:2 [ "identificador" => "sec0080" "titulo" => "TPPPS promoted mucosal and humoral immune responses induced by oral immunisation of the recombinant pNZ-ompA/L. lactis" ] 2 => array:2 [ "identificador" => "sec0085" "titulo" => "TPPPS promoted cell-mediated immune responses to the recombinant pNZ-ompA/L. lactis" ] 3 => array:2 [ "identificador" => "sec0090" "titulo" => "Inhibition of P. mirabilis colonisation and infection of BALB/c mice after vaccination with the recombinant pNZ-ompA/L. lactis" ] ] ] 5 => array:2 [ "identificador" => "sec0095" "titulo" => "Discussion" ] 6 => array:3 [ "identificador" => "sec0100" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0105" "titulo" => "Confidentiality of data" ] 1 => array:2 [ "identificador" => "sec0110" "titulo" => "Right to privacy and informed consent" ] 2 => array:2 [ "identificador" => "sec0115" "titulo" => "Protection of human subjects and animals in research" ] ] ] 7 => array:2 [ "identificador" => "sec0120" "titulo" => "Conflict of interest" ] 8 => array:2 [ "identificador" => "xack298749" "titulo" => "Acknowledgments" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2016-12-05" "fechaAceptado" => "2017-04-20" "PalabrasClave" => array:1 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec879393" "palabras" => array:5 [ 0 => "<span class="elsevierStyleItalic">Proteus mirabilis</span>" 1 => "ompA" 2 => "<span class="elsevierStyleItalic">Lactococcus lactis</span>" 3 => "Mucosal immune" 4 => "TPPPS" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleItalic">Proteus mirabilis</span> poses a critical burden on the breeding industry, but no efficient vaccine is available for animals.</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Method</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">A recombinant <span class="elsevierStyleItalic">Lactococcus lactis</span> expressing the ompA of <span class="elsevierStyleItalic">P. mirabilis</span> was used to develop a vaccine. The mucosal and systemic immune responses of the recombinant vaccine were evaluated in mice after oral immunisation. The inhibition on <span class="elsevierStyleItalic">P. mirabilis</span> colonisation of vaccines was also determined. Moreover, Taishan <span class="elsevierStyleItalic">Pinus massoniana</span> pollen polysaccharides (TPPPS) were used as adjuvants to examine the immunomodulatory effects.</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The pure recombinant <span class="elsevierStyleItalic">L. lactis</span> vaccine significantly induced the production of specific IgA and IgG, IL-2, IL-4, IFN-γ, and T lymphocyte proliferation, and the immunised mice exhibited significant resistance to <span class="elsevierStyleItalic">P. mirabilis</span> colonisation. Notably, the TPPPS adjuvant vaccines induced higher levels of immune responses than the pure <span class="elsevierStyleItalic">L. lactis</span>.</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">The <span class="elsevierStyleItalic">L. lactis</span> as a vaccine vehicle combined with TPPPS adjuvant provides a feasible method for preventing <span class="elsevierStyleItalic">P. mirabilis</span> infection.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Method" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] ] "multimedia" => array:6 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1573 "Ancho" => 2832 "Tamanyo" => 222118 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Identification of ompA gene expression in the recombinant <span class="elsevierStyleItalic">L. lactis</span>. (A) Identification of the recombinant pNZ-ompA plasmid by digestion with restriction enzymes. M: DNA 7000<span class="elsevierStyleHsp" style=""></span>bp ladder; 1: the recombinant pNZ-ompA plasmid digested with NcoI and XbaI. The digested <span class="elsevierStyleItalic">ompA</span> gene at the bottom (1107<span class="elsevierStyleHsp" style=""></span>bp); the backbone of the pNZ8149 plasmid at the top (2548<span class="elsevierStyleHsp" style=""></span>bp); 2: the recombinant pNZ-ompA plasmid digested by single NcoI enzyme (3655<span class="elsevierStyleHsp" style=""></span>bp); 3: the recombinant pNZ-ompA plasmid digested by single XbaI (3655<span class="elsevierStyleHsp" style=""></span>bp). (B) Phenotypic screening of recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> utilising lactose in Elliker medium. The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> presented yellow colony. (C) Detection of ompA expression by Western blot assay. The mouse anti-ompA monoclonal antibody was used in this assay. M: Pageruler pre-stained protein ladder; the predicted bands (approximately 45<span class="elsevierStyleHsp" style=""></span>kDa) of the ompA protein were detected in lanes 1–2. No band was shown in Lane 3 carrying a control protein β-actin.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 900 "Ancho" => 3339 "Tamanyo" => 106327 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Flow cytometric and immunofluorescence analysis of the <span class="elsevierStyleItalic">L. lactis</span> expressing ompA. (A) Flow cytometric analysis of the <span class="elsevierStyleItalic">L. lactis</span> expressing ompA. The mouse anti-ompA monoclonal antibody was used in this assay. The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> showed a significant increase of fluorescence intensity (green); the blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> showed negative fluorescence (red). (B and C) Immunofluorescence analysis of the <span class="elsevierStyleItalic">L. lactis</span> expressing ompA. The mouse anti-ompA monoclonal antibody was used in this assay. The recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span> showed positive green fluorescence on the cells (B). No fluorescence was shown in the blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span> (C).</p>" ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 3430 "Ancho" => 2496 "Tamanyo" => 348478 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Changes in the intestinal and tracheal specific sIgA and serum antibody titres of the mice inoculated with oral vaccines. Mice in six groups were inoculated with 50, 100, and 200<span class="elsevierStyleHsp" style=""></span>mg/mL of TPPPS adjuvant recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, pure recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span>, and PBS respectively at 0–4, 10–14, and 24–28<span class="elsevierStyleHsp" style=""></span>dpi. Intestinal and tracheal lavage fluids and serums were collected at 0, 14, 28, 42, and 56<span class="elsevierStyleHsp" style=""></span>dpi. The specific sIgA and serum antibody titres were then determined by indirect ELISA. All values shown are the means<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SD of three independent experiments. Different superscripts indicate a significant difference (<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05).</p>" ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 4301 "Ancho" => 2329 "Tamanyo" => 468126 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Changes in cytokines of the mice inoculated with vaccines. Mice in six groups were inoculated with 50, 100, and 200<span class="elsevierStyleHsp" style=""></span>mg/mL of TPPPS adjuvant recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, pure recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span>, and PBS respectively at 0–4, 10–14, and 24–28<span class="elsevierStyleHsp" style=""></span>dpi. Serum was collected at 0, 14, 28, 42, and 56<span class="elsevierStyleHsp" style=""></span>dpi. IL-2 (A), IFN-γ (B), IL-4 (C) and IL-10 (D) were detected by using the mouse IL-2, IFN-γ, IL-4 and IL-10 ELISA kits. All values shown are the means<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SD of three independent experiments. Different superscripts indicate a significant difference (<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05).</p>" ] ] 4 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 3454 "Ancho" => 2353 "Tamanyo" => 348992 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Changes in LTRs, CD4+, and CD8+ T lymphocytes in mice inoculated with vaccines. Mice in six groups were inoculated with 50, 100, and 200<span class="elsevierStyleHsp" style=""></span>mg/mL of TPPPS adjuvant recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, pure recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span>, and PBS respectively at 0–4, 10–14, and 24–28<span class="elsevierStyleHsp" style=""></span>dpi. Serum was collected at 0, 14, 28, 42, and 56<span class="elsevierStyleHsp" style=""></span>dpi. Then the LTRs, and the percentages of CD4+ and CD8+ T lymphocytes were detected by flow cytometry. An asterisk indicates that the value of the corresponding group was significantly different from that of the control group (<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.05).</p>" ] ] 5 => array:7 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 2558 "Ancho" => 2310 "Tamanyo" => 268761 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Intestinal colonisation and protective rates of <span class="elsevierStyleItalic">P. mirabilis</span>-challenged mice. Mice in six groups were inoculated with 50, 100, and 200<span class="elsevierStyleHsp" style=""></span>mg/mL of TPPPS adjuvant recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, pure recombinant pNZ-ompA/<span class="elsevierStyleItalic">L. lactis</span>, blank-pNZ/<span class="elsevierStyleItalic">L. lactis</span>, and PBS, respectively, at 0–4, 10–14, and 24–28<span class="elsevierStyleHsp" style=""></span>dpi. (A) At 29<span class="elsevierStyleHsp" style=""></span>dpi, 20 mice were challenged by oral inoculation of LD<span class="elsevierStyleInf">50</span> of <span class="elsevierStyleItalic">P. mirabilis</span>. Faecal shedding was monitored daily by determining the CFU of <span class="elsevierStyleItalic">P. mirabilis</span> in samples. (B) Two weeks after the last inoculation, 20 mice from each group were challenged with oral inoculation of 10 LD<span class="elsevierStyleInf">50</span> of <span class="elsevierStyleItalic">P. mirabilis</span>. The survival status of mice was calculated with the following formula: Survival rate (%)<span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>No. of surviving mice/Total No.<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>100.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:30 [ 0 => array:3 [ "identificador" => "bib0155" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Radial and spiral stream formation in <span class="elsevierStyleItalic">Proteus mirabilis</span> colonies" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:3 [ 0 => "C. Xue" 1 => "E.O. Budrene" 2 => "H.G. 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