Bacterial ghost: Future vaccine candidate for Aquaculture

 

 

B.T. Naveen Kumar¹, Babin Boppanna. K¹, Omkar V Byadgi¹ , Mansoor ahmad¹, Prabhugouda Shiriyappagouder¹, Pradeep L. doddamani², Kamalesh panda², Adil. B¹.

 

¹ Deparment of Aquaculture, ² Department of Fisheries Management, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University.

Corresponding author: naveenkumar504@gmail.com

 

Keywords:  Bacteria Ghost, Vaccine, Delivery vehicles, Fish immune system.

 

 

Rapid strides in aquaculture practices are achieved by geographical expansion and technological advancements in reproduction in captivity, larval rearing, formulated artificial diet and intensification in grow out systems. This intensification practice has given very good profit to the farmers and it led to intensification of larval rearing system with more technological advancement. These practices were reported to cause disease problems in the culture systems.

Disease control by the antibiotics in culture practices had reported to show improvement but residues of drugs in animal tissue, antibiotic resistance and transfer of antibiotic resistance gene has banned the farmers to use it (FAD). To overcome this problem, Vaccination was judged to be the best method.

The main purpose of vaccination is to enhance the immune system of an animal and recognize pathogenic agent to reduce the severity of a disease. This disease reduction or control in an animal is based on the recognition or interaction of immunoglobulin to vaccine agent (Inactive pathogen). Hence, involvement of specific immune system is major concern in the vaccination process.

Vaccination is done in three different methods

1.     Injection

2.     Immersion

3.     Oral vaccination

 Among these, oral vaccination is the better method because of its effectiveness and low cost. Oral vaccination technique like; killed pathogen, bacterial ghost, subunit, attenuated vaccine are in use at present. Among which, heat killed and attenuated vaccine often results altering the antigenic site or epitope (7). Though, the use of subunit vaccine is projected as substitute for the heat killed and attenuated vaccine and can be easily produced by expression of gene of interest in a host cell but its use in aquaculture is less it is mainly due to its poor immunogenic property and animals are not exposure to natural state of pathogenic part Hence, new approaches or methods should be developed to improve the vaccination in fish. The bacterial ghost system is one such vaccine.

 

What do you mean by Bacterial ghost (BGs)?

Bacterial ghost are mainly derived from gram negative bacteria (1). They are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structure. These are empty, non living envelope and retain surface antigenic protein structures in a natural state.

Fig.1: Bacterial ghost

 

Source:              http://2008.igem.org/Team:Slovenia/Results/Engineered_flagellin_vaccine/Engineered_bacterial_vaccine

 

Appearance of BGs: (Fig.1)

Ghost can be distinguished from their living unlysed cells using light microscopic examination by their: slight elongated appearance and transparent cells than living bacterial cell. It is provided with a tunnel (Hole- 40-200nm) at center or poles to expel the inner contents (Fig 2) (1).  

 

 

Fig. 2; High resolution field emission scanning electron micrograph of protein E-lysed Gram-negative bacteria. An arrow indicates the efflux of bacterial cytoplasm at the time point of lysis onset through the E-specific lysis tunnel.

Source: Ebensen et al., 2004 http://www.jimmunol.org/content/172/11/6858.full.html#ref-list-1

 

Properties of BGs:

BGs are free from the Cytoplasmic content and bacterial DNA and they are non living bacteria. The production process of BG does not denature the envelop part. Hence, all the antigenic determinants are preserved during production process of BG. In addition, this provides intrinsic adjuvant property to the animal and delivery system for drugs and DNA vaccine etc.

 

Production of BGs:

Bacterial ghost can be produced by expression of *gene E (bacteriophage phiX174) in a target cell (gram negative bacteria). It results in the lysis (Diameter 40-200nm) of bacterial cell. Where, this E- mediated lysis helps the bacterial to release all its  cytoplasmic contents to the environment, due to osmotic pressure difference between the cytoplasm and the surrounding medium (Fig. 2). Therefore, native structure of envelop remains intact and rigid. Thus, bacterial ghost is formed.

 

*Gene E

●    Gene E codes for a 91 aa polypeptide (1).

●    Gene E having a hydrophobic region at N terminal and end suggesting contranslational integration into the cytoplasmic membrane of bacterial (Gram negative bacteria) (1, 2).

●    Gene E expression and lysis of depends on the growth of the bacteria (gram negative bacteria) and its autolytic system (1).

 

 

Advantages:

 

●  BGs are not genetically modified organisms

●  Lyophilized BGs can be stored at ambient temperature

●  BGs are self adjuvant

●  There is no pathogenic threat to the animal

●  It can be produced easily by fermentation with bulk quantity

●  This is safe to use as vaccine candidate and delivery system

●  It is having a less cost of production 

●  Delivery vehicle for  DNA vaccine  (5)

●  Good recognition and uptake by antigen-presenting cells

●  BGs as micro-bioreactors for enzymatic reactions (1)

 

Application:

 

BGs as a vaccine:

BG of Edwardsiella tarda admistration to olive flounder (Paralichthys olivaceus) has found to be effective in preventing or protecting the fish from edwardsiellosis. The admistarion of BG via oral, immunization and immersion are effective in reducing the diseases in fish. In aquaculture practice, oral vaccine works better. Therefore, BG is the best vaccine for oral administration in aquaculture, due to its non living nature, stability, low cost of production, long shelf life. (9, 12)

BGs as adjuvant:

BGs production process does not denature the bacterial envelop or surface protein and preserve pathogen- associated molecular patterns (PAMPs) (1). These PAMPs induces the generation of cellular and humoral response in an experimental animal (3).

  

 BGs as antigen delivery vehicles:

The fusion of antigen to BGs through recombinant technology helps in delivery of antigen to target system (4). This fusion of antigen to membrane (BGs) did not affect the proper folding of enzymes. Hence, the Bacterial ghost is a novel vaccine delivery system and it provides excellent natural intrinsic adjuvant properties with versatile carrier functions for foreign antigens (6).

 

Conclusion:

Bacterial ghost is a novel vaccine candidate and a delivery system. It also provides natural adjuvant property, easy manufacturing, low production costs, and excellent safety profile. Hence, it is a promising technology for the development of more efficient vaccine in aquaculture practices.

 

References:

1.     Langemann T, Verena Juliana Koller, Abbas Muhammad, Pavol Kudela, Ulrike Beate Mayr and Werner Lubitz. The bacterial ghost platform system: Production and applications.  Bioengineered Bugs September/October 2010; 1:5, 326-336.

2.     Witte A, Lubitz W. Biochemical characterization of phiX174-protein-E-mediated lysis of Escherichia coli. Eur J Biochem 1989; 180:393-8.

3.     Riedmann EM, Kyd JM, Cripps AW, Lubitz W. Bacterial ghosts as adjuvant particles. Expert Rev Vaccines 2007; 6:241-53.

4.     Paukner S, Kohl G, Lubitz W. Bacterial ghosts as novel advanced drug delivery systems: antiproliferative activ ity of loaded doxorubicin in human Caco-2 cells. J Control Release 2004; 94:63-74.

5.     Ebensen T, Susanne Paukner, Claudia Link, Pavol Kudela, Carola de Domenico, Werner Lubitz, and Carlos A. Guzma«n. Bacterial Ghosts Are an Efficient Delivery System for DNA Vaccines. J Immunol 2004;172;6858-6865.

6.     Mayr UB, Petra Walcher, Chakameh Azimpour, Eva Riedmann, Christoph Haller, Werner Lubitz. Bacterial ghosts as antigen delivery vehicles. Advanced Drug Delivery Reviews 2005; 57; 1381Ð 1391.

7.     Szostak MP.  Andreas Hensel, Francis O. Eko, Reinhard Klein, Tatjana Auer, Horst Mader, Alexander Haslberger, Sebastian Bunka, Gerhard Wanner, Werner Lubitz. Bacterial ghosts: non-living candidate vaccines. Journal of Biotechnology 1996; 44; 161- 170.

8.     Se Ryaun Kwon, Yoon Kwon Nam, Sung Koo Kim, Ki Hong Kim. Protection of tilapia (oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts. Fish. Shellfish. Immunol.2006; 20: 621-626.

9.     Se Ryaun Kwon, Eun Hye Lee, Yoon Kwon Nam, Sung Koo Kim, Ki Hong Kim. Efficacy of oral immunization with Edwardsiella tarda ghost against edwardsiellosis in olive flounder (Paralichthys olivaceus). Aquaculture. 2007; 269: 84-88.

10.   Se Ryun Kwon,  Yue Jai Kang,  Dong Jin Lee, Eun Hye Lee, Yoon Kwon Nam, Sung Koo Kim, Ki Hong Kim. Generation of Vibrio anguillarum Ghost by Coexpression of PhiX 174 Lysis E gene and Staphylococcal Nuclease A Gene. Mo. Biotechnol. 2009; 42: 154-159.

11.   Ra, Chae-Hun, Yeong-Jin Kim, So-Jin Park, Chang-Wha Jeong, Yoon-Kwon Nam, Ki-Hong Kim, and Sung-Koo Kim. Evaluation of Optimal Culture Conditions for Recombinant Ghost Bacteria Vaccine Production with the Antigen of Streptococcus iniae GAPDH. J. Microbiol. Biotechnol. 2009; 19 (9): 982-986.

12.   Tu F.P., Chu W.H., Zhuang X.Y. and Lu C.P. Effect of oral immunization with Aeromonas hydrophilla ghosts on protection aganist experimental fish infection. Letters in Applied Microbiology. 2010; 50: 13-17.

 

 

 

S. N	Title	Outcome	Author	Journal	year
1	Protection of tilapia (oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts (ETG)	Fish immunized with ETG were found to be effective in controlling the fish from edwardsiellosis.	Se Ryaun Kwon, Yoon Kwon Nam, Sung Koo Kim, Ki Hong Kim	Fish and Shellfish Immunology	2006
2	Efficacy of oral immunization with Edwardsiella tarda ghost against edwardsiellosis in olive flounder (Paralichthys olivaceus)	ETG are reported to elicit immune response and protected the fishes from edwardsiellosis	Se Ryaun Kwon, Eun Hye Lee, Yoon Kwon Nam, Sung Koo Kim, Ki Hong Kim	Aquaculture	2007
3	Generation of Vibrio anguillarum Ghost by Coexpression of PhiX 174 Lysis E gene and Staphylococcal Nuclease A Gene	Developed a safety-enhanced Vibrio anguillarum ghost and it can be used as a vaccine in aquaculture.	Se Ryun Kwon,  Yue Jai Kang,  Dong Jin Lee, Eun Hye Lee, Yoon Kwon Nam, Sung Koo Kim, Ki Hong Kim	Molecular Biotechnology	2009
4	Evaluation of Optimal Culture Conditions for Recombinant Ghost Bacteria Vaccine Production with the Antigen of Streptococcus iniae GAPDH	It showed higher survival rate and good antibodies titre than formalin killed cell vaccine.	Ra, Chae-Hun, Yeong-Jin Kim, So-Jin Park, Chang-Wha Jeong, Yoon-Kwon Nam, Ki-Hong Kim, and Sung-Koo Kim.	Journal of Microbiology and Biotechnology	2009
5	Effect of oral immunization with Aeromonas hydrophilla ghosts on protection aganist experimental fish infection	Higher protection to Aeromonas hydrophilla infection in Carasius arraus gibilla by increased antibody titre.	F.P. Tu, W.H. Chu, X.Y. Zhuang and C.P. Lu	Letters in Applied Microbiology	2010

TABLE: STUDIES CARRIED OUT IN FISHERIES