Vaccine Research
Volume:3 Issue: 2, Summer and Autumn 2016

Mucosal surfaces of the body provide a universal entry portal for all known and emerging infectious pathogenic microbes. Therefore, it seems that special vaccination strategies are needed for vaccines that can hinder the entry capability of pathogenic microbes through the mucosal surfaces. Lactic acid bacteria are widely used in the food industry and are presently applied as delivery vehicles in many biological investigations. Among these bacteria, Lactococcus lactis is considered as a promising candidate for mucosal live vaccines to be used as an antigen delivery vector. This is an attractive alternative and a safer vaccination strategy against the pathogens, compared to other conventional methods. In this review, we summarized the applications of L. lactis as a mucosal vector of vaccine delivery for heterologous expression of proteins and its applications in biotechnology.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of livestock that is categorized in list A of animal diseases by the World Organisation for Animal Health (OIE). Vaccination is effective against FMD and the vaccine production centers largely use the industrial ultra-filtration and chromatography in order to remove the cellular proteins as well as the non-structural viral proteins. The recommended method for the purification and quantification of the active ingredient of vaccines is 140S quantitative sucrose density gradient analysis. Despite many advantages, this method is highly operator-dependent and is not suitable for large-scale vaccine productions. The main objective of this study was to compare and evaluate two chromatography resins (i.e. Sephacryl S-300 and Sephacryl S-500) to separate FMD virus particles from the non-structural viral proteins.
The resins were compared for gel filtration chromatography and the virus infectivity titration (CCID50% / ml) and real-time PCR amplification analyses were performed.
The results indicated that Sephacryl S-500 was not able to separate blue dextran from bovine serum albumin; therefore, it was not suitable for separation of the whole virus from the non-structural proteins while Sephacryl S-300 was suitable for this purpose.
Sephacryl S-300 is a suitable resin for preparation of purified virus for large-scale FMD vaccine production.

Influenza A virus causes unpredictable epidemics and pandemics by creating antigenic variations. With the appearance of each new strain, rapid emergency countermeasures are taken against this new strain. Hence, designing an applicable and cross protective strategy to counter this virus is of great importance. To achieve this, choosing conserved antigenic regions in influenza virus proteins for making a universal vaccine is one of the best options. M2 channel in influenza virus membrane has a conserved sequence in ectodomain region called M2e. This region is the same between the majority of influenza A virus strains. But this peptide region is not a good stimulant for the immune system due to its short length. Immunoadjuvant property of Salmonella Typhimurium flagellin (FliC) has been previously shown to arouse immunity in the mucosal region. A combination of M2e and FliC was used in a construct in this study.
To increase the possibility of immunugencity of this construct, in silico predictions of this fusion peptide construct were performed first. Three repeats of M2e gene and FliC sequence were cloned into pET28 vector and then were sub-cloned to pHT43. Finally, the construct was transformed into Bacillus subtilis by electroporation. After IPTG induction, the total cell protein and the supernatant protein were analyzed via protein analyses methods.
Based on in silico immunogenicity results, the designed recombinant peptide was deemed suitable to be used as a universal influenza vaccine candidate. The validity of pHT43.3M2e.FliC construct was confirmed by restriction map analysis. Western-blotting confirmed the presence of recombinant protein.
The fusion peptide produced in this research, is the first step in designing a universal influenza vaccine which needs to be assessed in animal models alongside proper control groups in future studies.

Polyclonal antibodies are required to be affinity purified. Improved purification methods of polyclonal antibody provide an opportunity to pick the most purified immunoglobulins as a primary or secondary antibody in immunoassays that are included in many vaccine studies. Two common techniques for purifying proteins is salt precipitation and chromatography purification. Our work focuses on purification of polyclonal antibodies against recombinant human erythropoietin (EPO) antigen using these techniques.
A polyclonal antibody was produced by antigen injection with Freund's adjuvant into female albino rabbits. After separation of immunoglobulins using caprylic acid and ammonium sulfate precipitation, selected samples were analyzed by ion exchange chromatography for separation of polyclonal antibody from albumin. The purified proteins were analyzed by SDS-PAGE and antibody was detected by Western Blot analysis and ELISA. Results of immunodiffusion test detected polyclonal antibody production in rabbits.
Caprylic acid precipitation was shown to be a more effective purification method than ammonium sulfate. Analysis of protein by spectrophotometer showed 97.6% purity by caprylic acid and 77% purity by ammonium sulfate method. Western Blot and ELISA tests confirmed the presence of antibody against EPO.
These findings suggest that caprylic acid can be used as a quality control method in a production facility with minimal cost. On the other hand, ion exchange chromatography is the most common purification method for proteins. Therefore, combination of these techniques may effectively reduce contaminations in antibody purification procedures which may positively affect the interpretations of vaccine efficacies.

Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in human. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic Porin. This study aimed to clone and determine the expression of PorA as the first step for producing a proper antigen in a vaccine study against N. meningitidis.
An approximately 1200-bp fragment of porA gene was amplified by PCR using N. meningitidis serogroup A genomic DNA and then cloned into prokaryotic expression vector pET-28a. The resulting construct (pET28a-porA plasmid) was transformed into competent E.coli BL21 cells for expression of recombinant protein. The proper overexpression of the recombinant protein was verified by SDS-PAGE and Western Blotting.
Cloning of porA was confirmed by colony-PCR and enzymatic digestion. The nucleotide sequence homology of the cloned porA gene was 97% , compared to the reference gene (NCBI GenBank accession number AL157959.1). The prokaryotic expression system (pET28a-porA- BL21) could produce our 45-kDa target recombinant protein, efficiently.
The prokaryotic expression system and conditions used in this study provides an applicable method for producing recombinant PorA and possibly many other bacterial outer membrane proteins for future vaccine studies.

During the production process of vaccines against viral diseases, virus inactivation plays a crucial role. β-Propiolactone (BPL) is a compound that is frequently used for inactivation of viruses and production of the vaccines against viral diseases such as rabies. In this study, the effects of concentration of BPL as well as the duration and temperature of incubation with BPL on the infectivity of the rabies virus was investigated.
Fixed rabies (Pasteur Virus) PV strain grown on BHK21C13 cell was treated with 1:3000, 1:4000 and 1:5000 final concentrations of BPL at 4°C, 21°C and 37°C and their effects were evaluated after 24, 48, 72 h and 5 days. Each sample was titrated after various intervals and tested for the viral infectivity in BSR cells. The BSR cells were infected by triple serial dilutions of the rabies virus PV strain, and then infectivity was detected by the fluorescent technique.
Entire loss of infectivity of the virus treated at 4°C with 1:3000 and 1:4000 of BPL was evident at the end of the 24 and 48 h incubation periods, respectively. Moreover, the loss of infectivity of the virus treated with all tested concentrations of BPL was evident at the end of the 72 h at 21°C and 4°C.
These findings suggest that aside from BPL concentration, temperature plays the most important role on BPL inactivation. The inactivation effect could be observed at the end of 72 h incubation period; however, the best economic condition for rabies virus inactivation was shown to be 1:4000 at 4°C for 48 h.

Pathogenic strains of Proteus mirabilis have important roles in urinary tract infection. Proteus toxic agglutinin (Pta) is amongst the most important virulence factors of P. mirabilis. This protein has a conserved sequence present in all the strains which could be evaluated as a novel vaccine target against them. The aims of the current study were the expression, purification and characterization of a truncated Pta protein of P. mirabilis strain HI4320 as well as the bioinformatics analysis of the truncated protein.
The passenger domain of pta genes in P. mirabilis was evaluated by bioinformatics studies. The selected domain (residues 207-730) was amplified by PCR and cloned into pET28a expression vector. The Pta was expressed in BL21 (DE3) host and purified by Ni-NTA resin. The analyses of the purified protein were performed by SDS-PAGE and Western blotting.
The bioinformatics studies predicted the appropriateness of the passenger domain of Pta protein in terms of conservation, stability and cell-surface exposure. The length of PCR fragment of truncated form of pta gene was ~1500 bp. The cloning and expression of the truncated pta gene was successfully performed using pET28a-BL21 (DE3) system. Analyses of the purified Pta by SDS-PAGE and Western blotting confirmed the purification of a ~60 kDa His-tagged polypeptide.
The high frequency of P. mirabilis infection, especially in patients with abnormalities in their urinary tracts and also the rising of antibiotic resistance among the strains of this pathogen point to the need for effective controlling measures against them. In this regard, the passenger domain of Pta could be considered as a vaccine target. The efficacy and in-vivo immunogenicity of this purified protein is currently under study.