Evaluation of in Ovo Vaccination of DNA Vaccines for Campylobacter Control in Broiler Chickens ⇑ Xiang Liu, Lindsay Jones Adams, Ximin Zeng, Jun Lin
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Vaccine 37 (2019) 3785–3792 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Evaluation of in ovo vaccination of DNA vaccines for Campylobacter control in broiler chickens ⇑ Xiang Liu, Lindsay Jones Adams, Ximin Zeng, Jun Lin Department of Animal Science, The University of Tennessee, 2506 River Drive, Knoxville, TN 37996, USA article info abstract Article history: Campylobacter is the leading bacterial cause of human enteritis in developed countries. Chicken is a major Received 7 August 2018 natural host of Campylobacter. Thus, on-farm control of Campylobacter load in poultry would reduce the Received in revised form 13 May 2019 risk of human exposure to this pathogen. Vaccination is an attractive intervention measure to mitigate Accepted 20 May 2019 Campylobacter in poultry. Our previous studies have demonstrated that Campylobacter outer membrane Available online 3 June 2019 proteins CmeC (a component of multidrug efflux pump) and CfrA (ferric enterobactin receptor) are fea- sible and promising candidates for vaccine development. In this study, by targeting these two attractive Keywords: vaccine candidates, we explored and evaluated a new vaccination strategy, which combines the in ovo Campylobacter jejuni vaccination route and novel DNA vaccine formulation, for Campylobacter control in broilers. We observed DNA vaccine In ovo vaccination that direct cloning of cfrA or cmeC gene into the eukaryotic expression vector pCAGGS did not lead to suf- Poultry ficient level of production of the target proteins in the eukaryotic HEK-293 cell line. However, introduc- Kozak sequence tion of the Kozak consensus sequence (ACCATGG) in the cloned bacterial genes greatly enhanced production of inserted gene in eukaryotic cells, creating desired DNA vaccines. Subsequently, the vali- dated DNA vaccines were prepared and used for two independent in ovo vaccination trials to evaluate their immune response and protective efficacy. However, single in ovo injection of specific DNA vaccine at 18th day of embryonation, regardless using neutral lipid-protected vector or not, failed to trigger sig- nificant IgG and IgA immune responses and did not confer protection against C. jejuni colonization in the intestine of chickens. In conclusion, this study demonstrates that the Kozak sequence is critically impor- tant for construction of the DNA vaccine expressing prokaryotic gene. The optimal regimen for in ovo vac- cination of DNA vaccine for Campylobacter control in poultry needs to be determined in future studies. Ó 2019 Elsevier Ltd. All rights reserved. 1. Introduction practical vaccine available to control Campylobacter infections in poultry, primarily due to lack of effective and practical vaccination Thermophilic Campylobacter species including Campylobacter strategies [6,7]. jejuni and Campylobacter coli are one of the most commonly recog- To achieve the goal of developing effective, safe, inexpensive, nized bacterial causes of foodborne illnesses in the United States and convenient vaccination strategies that could be practically and other developed countries [1]. This pathogenic organism used to in poultry for mitigation of C. jejuni, our laboratory has causes watery diarrhea, fever, and abdominal cramping in patients made significant progress in the past years to identify conserved and is unusually associated with Guillain-Barre Syndrome, an protective antigens in C. jejuni, a paramount and critical step acute flaccid muscular paralysis that may result in respiratory towards the design of effective vaccines against Campylobacter. muscle compromise and death [2,3]. Poultry is considered one of Specifically, we have identified and characterized two surface- the major reservoir of Campylobacter and therefore the main source exposed proteins, CmeC and CfrA, that play an essential role in C. of human campylobacteriosis [4,5]. On-farm control of Campy- jejuni colonization in the chicken intestine [8–13]. CfrA is a lobacter in poultry would therefore reduce the risk of human surface-exposed ‘gatekeeper’ that is essential for C. jejuni coloniza- campylobacteriosis and have a significant impact on food safety tion by mediating ferric enterobactin high affinity iron acquisition and public health. However, to date, there is still no effective and [11]. CmeC is an essential outer membrane protein component of CmeABC multidrug efflux that plays a critical role in multidrug resistance and C. jejuni colonization [8,9,12,13]. The following find- ⇑ Corresponding author at: Department of Animal Science, The University of Tennessee, 2506 River Drive, Knoxville, TN 37996-4574, USA. ings from our previous studies provided compelling evidence that E-mail address: [email protected] (J. Lin). both CmeC and CfrA have significant advantages compared to other https://doi.org/10.1016/j.vaccine.2019.05.082 0264-410X/Ó 2019 Elsevier Ltd. All rights reserved. 3786 X. Liu et al. / Vaccine 37 (2019) 3785–3792 immunogenic/protective antigens identified in C. jejuni: (1) both England). The E. coli strains were grown on Luria-Bertani (LB, BD CfrA and CmeC are immunogenic in poultry and elicit a specific Difco) plates containing 50 lg/mL of ampicillin (Amp) or in LB antibody response during C. jejuni infection in poultry [9–11]; (2) broth containing 50 lg/mL of Amp with shaking (250 rpm) at CfrA and CmeC specific antibodies greatly inhibited the function 37 °C overnight. of corresponding target and significantly reduced growth of C. jejuni [11,12]; (3) both CfrA and CmeC are prevalent and highly 2.2. PCR conserved in diverse C. jejuni strains [9,11,12]; (4) CfrA and CmeC are highly induced and produced in the intestinal tract [9–12]; Primers used in this study and the expected sizes of the prod- and (5) inhibition of CmeABC efflux pump by a pump inhibitor ucts are listed in Table 2. Each PCR was performed with a 50 lL increased susceptibility of C. jejuni to multiple antimicrobials and mixture containing 200 lM deoxynucleoside triphosphates, reduced in vivo colonization of C. jejuni in chickens [13]. Clearly, 200 nM of each primer, 50 ng of JL241 template DNA, 2.5 mM these comprehensive molecular, immunogenic, functional studies MgSO4, and 5 U PfuUltra II high-fidelity DNA Polymerase (Strata- have shown that CmeC and CfrA are promising candidates for gene). The temperature-cycling parameters are typically as fol- developing an effective vaccine against C. jejuni in poultry. lows: 95 °C for 5 min for denaturation, 32 cycles of 1 min at Vaccination at embryonation day 18 has proven to be a safe, 94 °C, 1 min at 58 °C, 90 sec at 72 °C, and a final extension step effective, and convenient method for vaccination of chickens of 45 sec at 72 °C, though cycling conditions slightly varied accord- against viral, bacterial and protozoal diseases [14]. In addition, ing to estimated product sizes (1 min per kb for extension at 72 °C). DNA vaccine, the eukaryotic expression plasmid bearing the gene PCR products were further purified with the QIAquick Purification of desired target antigen, has been successfully developed to pre- Kit (Qiagen) for cloning procedures or sequencing analysis. vent and control infectious diseases and other disease in human and animals [15,16]. DNA vaccination has various advantages, 2.3. Construction of DNA vaccines. including the ability to induce both cellular and humoral immune response, lack of risk for infection, long-term persistence of The pCAGGS vector (Table 1) is a eukaryotic expression vector immunogen, easiness of manufacture, and stability for shipping containing the chicken b-actin promoter, the CMV immediately and storage [16,17]. More importantly, when combined with in early enhancer (CMV-IE), the SV40 origin of replication (SV40 ovo vaccination, DNA vaccination could offer various advantages OriC), and the ampicillin resistance cassette (Ampr) for selection for poultry [18]. Therefore, by targeting the two attractive vaccine (a kind gift from Dr. Miyazaki, University of Tokyo, Japan) [19]. candidates CfrA and CmeC, in this study, we explored and evalu- The full-length cfrA and cmeC fragments from C. jejuni NCTC ated the immunogenicity and protective efficacy of a new vaccina- 11168 were first PCR amplified using primer pairs of pCAGGS_C- tion strategy, which includes the in ovo vaccination route and DNA frA_F/pCAGGS_CfrA_R and pCAGGS_CmeC_F/pCAGGS_CmeC_R, vaccine formulation, for Campylobacter control in broilers. respectively (Table 2). All these primers have a XhoI site at the 50 end. The PCR products were digested by XhoI and ligated into 2. Materials and methods pCAGGS, which previously has been digested with the same enzyme. The ligation mixture was introduced into Top10 cells. 2.1. Bacterial strains and growth conditions Transformants were selected on LB agar plates containing ampi- cillin. The plasmids from randomly selected transformants were The major bacterial strains and plasmids used in this study, and extracted and analyzed by agarose gel electrophoresis. The recom- their sources, are listed in Table 1. The standard Campylobacter binant plasmids with insertion were further subjected to PCR jejuni NCTC 11168 (JL241) was used for amplification of cfrA and screening using vector-specific primer together with inserted cmeC genes. JL241 was routinely grown in Mueller Hinton (MH) gene-specific primer for identification of the recombinant plasmids broth (BD Difco, Sparks, MD) or on MH agar plates under microaer- with correct orientation of specific inserted gene. The identified ophilic conditions (5% O2, 10% CO2, 85% N2)at42°C.