Background
Clostridium difficile is one of the most important pathogens in nosocomial infections.
C. difficile infection (CDI) causes 10–20% of antibiotic-associated diarrhea, 75% of antibiotic-associated colitis, and nearly 100% of pseudomembranous colitis in hospitals (referred to as
C. difficile-associated diseases, CDADs), leading to billions of dollars in economic losses worldwide every year [
1]. In general, CDADs occur mainly in people with long-term use of antibiotics, the use of anticancer drugs, long-term hospitalization or immune defects, especially those with a decline in immune function or the elderly [
1]. With the development of medical industry and the increasing use of antibiotics, the rate of CDI has been substantially increased in China [
2]. Effective strategies are urgently needed for CDI prevention in the high-risk population of CDADs.
Due to antibiotic resistance and inherent physiological factors of the pathogen, antibiotic treatment of CDI can be challenging while oral immunization with vaccines is generally considered to be an important pathway for CDI prevention [
1]. Vaccine development involves the establishment of an appropriate animal model and further evaluation of the efficacy and safety of the vaccine. In previous research of
C. difficile vaccines, Kink
et al.[
3] successfully established CDI model in hamster by intragastric gavage of 10
5CFU
C. difficile 18–24 h after subcutaneous injection of clindamycin (CLDM, 1 mg per 100 g body weight), and Torres
et al. [
4] administered the animals with 10
5CFU
C. difficile 3-h after CLDM gavage (0.5 mg per 100 g body weight). In general, golden hamster is considered ideal for establishing CDI model, because
C. difficile-produced toxins can be neutralized by anti-
C. difficile antibodies and CLDM-induced colitis model can be used as animal model of human CDIs.
Regarding the development of vaccines for
C. difficile, great research progress has been made over the last two decades [
1]. For example, Torres
et al.[
4] reported that formalin-inactivated
C. difficile culture filtrate has a protective effect on hamsters by nasal, peritoneal, or subcutaneous administration. Ryan
et al.[
5] and Ward
et al.[
6] developed recombinant vaccines for
C. difficile by engineering a plasmid to express recombinant toxin A proteins from the nontoxic C-terminal receptor binding region of
C. difficile toxin A (TcdA) covalently bonded to polysaccharide of other bacterium, and then introducing this plasmid into attenuated
Salmonella typhimuriu or
Vibrio cholerae. However, the application of previously developed vaccines for
C. difficile has been limited for various reasons: 1) Vaccines for passive immunization of CDI are thought expensive and inconvenient in storage and transport; 2) Attenuated vaccines are commonly treated with formalin to inactivate the antigen or given with adjuvant, sometimes through invasive routes of immunization such as subcutaneous and intraperitoneal injection, thus are not easily accepted by the patients. 3) Recombinant vaccines that are carried by attenuated
S. typhimuriu or
V. cholerae are of concern in terms of biosafety [
1].
Lactococcus lactis is a harmless food industry bacterium that has been used extensively for producing a variety of peptides, proteins, and oral vaccines. As compared to the vaccine carrier
E. coli,
L. lactis can be a superior alternative because it produces less protease with no endotoxin [
7]. In the literature, Dieye
et al. [
8] designed a protein-targeting system for lactic acid bacteria and found that the
L. lactis expression system constructed with the P59 promoter and Usp45 single peptide (SPUsp45) was capable of expression and extracellular secretion of target nuclease while the expression system constructed with P59, Usp45, and the cell wall-anchored sequence of protein M6 (cwaM6) was capable of extracellular secretion of the nuclease as well as anchoring it onto the cell wall of
L. lactis and
Bacterium lacticum. In a following study, Ribeiro
et al.[
9] expressed a fusion protein containing the
Brucella abortus antigen L7/L12 and the
Streptococcus pyogenes cwaM6 in
L. lactis, which allowed the antigen anchored on the cell surface, thus improving its antigenicity. These authors further developed a food-grade live vaccine for immunization of
B. abortus, which showed a protective effect on mice under laboratory conditions [
9]. In addition, Mannam
et al.[
10] made a mucosal vaccine from live, recombinant
L. lactis, which protected mice against pharyngeal infection with
S. pyogenes. Together these researches have provided a completely new direction for development of vaccines, especially live probiotic preparations for oral immunization.
Despite potential advantages of
L. lactis as the carrier of live vaccine, no studies have been reported on oral immunization with
L. lactis live vaccines for preventing CDI till date. Whether vaccines made from live, recombinant
L. lactis are effective for preventing CDI remains unclear. Previously, we constructed a gene expression system in
L. lactis based on the work by Dieye
et al.[
8]. In the present study, we modified the gene expression system to develop recombinant
L. lactis live vaccine for
C. difficile, and then vaccinated a CDI animal model of golden hamsters by oral immunization. Pathological and immunological parameters of the animals were assayed under laboratory conditions. Results were used to evaluate the effect of recombinant
L. lactis live vaccine for preventing CDI.
Competing interests
There is no financial competing interest to declare in relation to this manuscript.
Authors’ contributions
XQC designed the research. XQY performed the research, analyzed data, and wrote the paper. YGZ, BJ, DY analyzed data, edited the manuscript. All authors read and approved the final manuscript.