Protection against tetanus toxin after intragastric administration of two recombinant lactic acid bacteria: impact of strain viability and in vivo persistence
Introduction
The potential of live recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the mucosal immune system has been investigated during the last decade [1], [2], [3], [4], [5], [6], [7]. This approach offers a number of advantages over the traditional parenteral vaccination, such as non-invasiveness and the possibility to elicit both systemic and mucosal immune responses. In addition, these Gram-positive non-pathogenic bacterial vectors are generally regarded as safe [8]. They represent an original alternative to the use of attenuated pathogenic bacterial carriers such as Salmonella, Bordetella, Vibrio and Mycobacterium [9], [10], [11], [12], [13], [14]. The LAB are mostly known for their widespread use as starter strains in the food and feed industry, but also for the probiotic effects that certain species or strains may exert in humans or animals [15], [16]. Several LAB strains are quite acid resistant and should thus be able to effectively survive passage through the stomach. This property is however species- and strain-dependent. For example, Lactobacillus plantarum NCIMB8826 exhibited a higher and longer survival in the human gastro-intestinal tract than Lactococcus lactis MG1363, when fed to healthy subjects [17]. The authors concluded that the pharmacokinetic properties of Lb. plantarum are promising for its development as vaccine vehicle. In addition, given LAB strains, mostly belonging to the Lactobacillus genus, are able to persist in mucosal cavities such as the mouth, the urogenital or the gastro-intestinal tract. Their development as vaccine has included the use of both non-colonizing (Lc. lactis) [1], [2] and colonizing (S. gordonii, Lactobacillus spp.) [3], [4], [5], [6], [7], [18] strains. A number of antigens have been efficiently produced in both systems, but the most complete immunological studies have been performed with the tetanus toxin fragment C (TTFC) as model antigen. Recombinant strains producing this model antigen were shown to induce high protective local and systemic antibody responses as well as cellular immune responses, after parenteral and intranasal immunizations [3], [4], [5], [6], [7], [18]. While both gut colonizers and non-colonizers seem to work equally well by these routes, the importance of colonization or persistence in oral administration remains poorly investigated. To address this question, we have undertaken a comparative study of the immunogenicity of recombinant lactobacilli (Lb. plantarum) and lactococci (Lc. lactis) producing equivalent levels of TTFC, by oral (intragastric) immunization. Moreover, the immune responses elicited by live and inactivated recombinant LAB were compared.
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Bacterial strains and culture conditions
All LAB strains used in this study are listed in Table 1. The recombinant strain of Lc. lactis MG1363 synthesizing cytoplasmic TTFC was constructed using the replicative pMEC46 plasmid in which the heterologous gene is under the control of the nisin (pnisA) inducible promoter as previously described [19], [20]. Lb. plantarum NCIMB8826 and Lc. lactis MG1363 strains harboring the cloning vectors pTG2247 [19] and pTX [1], respectively, were used as control strains.
Lb. plantarum was grown at 37 °C
TTFC production by the recombinant Lb. plantarum and Lc. lactis strains
To estimate the TTFC amount contained in the different inocula, cells extracts corresponding to 1:10 of the bacterial dose (108 cfu) were analyzed by immunoblotting. As shown in Fig. 1, a specific signal at the expected molecular mass (47 kDa) was detected for both TTFC-producing strains. As previously described [7], TTFC production was notably higher in the Lb. plantarum NCIMB8826 (pMEC127) than in the NCIMB8826 (pMEC4) strain. The level of antigen production was found similar and very high
Discussion
Recombinant lactobacilli were previously shown to induce both systemic and local immune responses after intranasal immunization [6]. In a limited number of experiments, these results were extended to the intragastric route [5], [7]. In the present work, we evaluated the impact of the persistence capacity of two lactic acid bacterial vectors on the immunogenicity and protective activity after intragastric administration. To address this point, we compared the strain Lb. plantarum NCIMB8826 as a
Acknowledgements
This work was supported by the EU BIO4-CT96-0542 grant, the Institut Pasteur de Lille, the Institut Pasteur de Bruxelles and FEDER funds. We are grateful to E. Van Nerom and F. Tweepenninckx for their skillful help with protection experiments. We very much appreciated the stimulating discussions with J. Delcour, P. Hols, and J.M. Wells. Rabbit anti-TTFC antibodies were kindly supplied by Dr. E. Sablon, Innogenetics N.V., Belgium.
References (22)
- et al.
Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C
Vaccine
(1997) - et al.
Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice
Vaccine
(1997) - et al.
Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice
Vaccine
(2001) - et al.
Comparison of the immune responses induced by local immunizations with recombinant Lactobacillus plantarum producing tetanus toxin fragment C in different cellular locations
Vaccine
(2002) - et al.
Potential of using lactic acid bacteria for therapy and immunomodulation in man
FEMS Microbiol. Rev.
(1993) - et al.
Overview of gut flora and probiotics
Int. J. Food Microbiol.
(1998) Immunomodulation by treatment with Lactobacillus casei strain Shirota
Int. J. Food Microbiol.
(1998)- et al.
Oral vaccination of mice against tetanus with recombinant Lactococcus lactis
Nat. Biotech.
(1997) - et al.
Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli
Immunology
(2000) - et al.
Mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant Lactobacillus plantarum
Infect. Immun.
(2001)
Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus toxin vaccine
Biotechnology
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2016, Infection, Genetics and EvolutionCitation Excerpt :Different bacteria have been used such as L. casei (Song et al., 2014; Li et al., 2009), Salmonella enterica (Kulkarni et al., 2008, 2010; Zekarias et al., 2008), and Bacillus subtilis (Hoang et al., 2008) as the antigen delivery systems. L. casei has the potential immune-modulatory properties and is widely used for expressing several heterologous antigens as vaccines in animal models with promising results (Campos et al., 2008; Maassen et al., 1999; Adachi et al., 2010; Ferreira et al., 2009; Grangette et al., 2002; Lee et al., 2006; Oliveira et al., 2006; Wen et al., 2012; Yoon et al., 2012). To our best knowledge, the current study is the first description for showing an effective immunity response for ε-toxoid of C. perfringens following oral administrative of this L. casei-based vaccine.