Efficacy of a Eudragit L30D-55 encapsulated oral vaccine containing inactivated bacteria (Lactococcus garvieae/Streptococcus iniae) in rainbow trout (Oncorhynchus mykiss).

The efficacy of a Eudragit L30D-55 encapsulated vaccine against Lactococcus garvieae and Streptococcus iniae was investigated in rainbow trout. Fish were divided into four groups and fed the different experimental feeds. Groups were: A) fish immunized by Eudragit-coated pellets containing vaccine, B) fish immunized by vaccine-coated pellets without Eudragit, C) fish fed Eudragit-coated pellets without vaccine and D) fish fed pellets without vaccine orEudragit (control group). In groups A and B, the vaccination was conducted for 14 days. Similar to groups A and B, fish of group C were fed 14 days with pellets coated with Eudragit and afterwards they were fed control diet. Serum samples were taken on day 0, 20, 40 and 60 of the experiment. After 60 days, fish were challenged with L. garvieae and S. iniae. In almost all groups, innate immunity components including alternative complement activity, lysozyme activity, bactericidal activity, IgM and total protein showed no significant changes during the 60 days that the experiment lasted. However, the blood respiratory burst activity and lysozyme activity showed a significant increase on day 20 of experiment in groups B and D respectively (P < 0.05). The relative expression of immune-related genes including IL-6 and IgM genes was higher in vaccinated fish, with the highest expression in those immunized by Eudragit-coated pellets (Group A). In addition, the relative expression of IL-6 and IgM peaked on day 20 but decreased on day 60 in vaccinated groups. The ELISA antibody titer against L. garvieae increased from day 20 and peaked on day 60 of experiment (P < 0.05). Also, the antibody titer against L. garvieae was higher in fish immunized by Eudragit-coated pellets (Group A) compared to fish of group C and control. After bacterial challenge, a survival percentages of % 85 ±â€¯7.07% (challenged with S. iniae) and % 72.21 ±â€¯7.8% (challenged with L. garvieae) were observed respectively in groups immunized with pellets coated with Eudragit L30D-55 (Group A), which were higher than survival percentages obtained in other experimental groups (P < 0.05). The results of the present study demonstrate that the oral administration of Eudragit L30D-55-encapsulated vaccine appropriately protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines.

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine ß-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials.

Mice immunization with live lactococci displaying a surface anchored HPV-16 E7 oncoprotein.

E7 oncoprotein of human papillomavirus-16 (HPV-16) is constitutively produced in cervical cancer (CxCa) and is a good candidate for the design of therapeutic vaccines. In this work, the nisin-controlled expression system was used to display the E7 protein at the cell surface of the food-grade Gram-positive bacterium Lactococcus lactis. An efficient cell wall anchoring of E7 was obtained. Intranasal administration of these recombinant lactococci in mice induced an HPV-16 E7-specific immune response. This is the first report of E7 cell wall anchoring in L. lactis and represents one more step towards the use of live food-grade bacteria to fight against CxCa.

Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited culture conditions.

Infection by Lactococcus garvieae has become a widely recognised problem associated with intensively cultured fish. Long-term control of fish infections may be possible by vaccination providing a suitable and efficacious epitope is expressed during production of cells used for vaccine preparation. The identification of novel vaccine candidates must, therefore, consider how the host species recognises and responds to bacterial cell components. L. garvieae was cultured in iron deficient, limited and haem iron enriched media and the whole cell proteins expressed under these conditions were compared with those expressed in bacteria extracted with Percoll gradients directly from spleen tissue of infected rainbow trout (Oncorhynchus mykiss). SDS-PAGE of the cell proteins showed the existence of several different electropherotypes according to the iron status of the culture media. Only minor differences in cell protein profile were detected in bacteria obtained directly from fish spleens, but when the electropherograms were analysed by Western blots using L. garvieae hyperimmune fish sera, several proteins could be identified that were expressed only when L. garvieae was growing in vivo. Siderophore could be detected in culture supernatant of iron deficient, limited and haem iron enriched media but not in media with higher nutrient concentrations. The siderophore could not be identified as a type of catechol or hydroxymate. Rainbow trout recognise proteins in the range of approximately 50-80 kDa for bacterial cells obtained without subculture from infected fish and culture conditions can influence protein profiles for this pathogen.

Antibody increases phagocytosis and killing of Lactococcus garvieae by rainbow trout (Oncorhynchus mykiss, L.) macrophages.

The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.

Production of human tumor necrosis factor alpha, interleukin-6, and interleukin-10 is induced by lactic acid bacteria.

To investigate the role of cytokines in interactions between lactic acid bacteria and the immune system, we measured production of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 from human peripheral blood mononuclear cells after stimulation with live or glutaraldehyde-fixed bacteria. Production of tumor necrosis factor alpha, IL-6, and, in some cases, IL-10 was induced in amounts even greater than those obtained with lipopolysaccharide as a stimulant. Our results suggest that lactic acid bacteria can stimulate nonspecific immunity.