Vaccine Research
Volume:4 Issue: 2, Summer and Autumn 2017

Human papillomavirus (HPV) is the main causative agent of cervical cancer worldwide leading to a big health problem, especially in the developing countries. Among 14 common high-risk genotypes, HPV16 accounts for more than 50% of all cervical cancers. The current prophylactic vaccines against HPV infection are based on L1 protein. Due to some drawbacks in the current vaccines such as their restrictions to genotype and relatively high costs, second-generation HPV vaccines, based on L2 protein, are under development. The evaluation of the neutralization efficiency of the antibodies against the virus needs pseudoviruses consisting of L1 and L2 capsid proteins harboring a reporter gene. The aim of this study was assembling HPV16 L1 and L2 proteins into a pseudovirion that contains a reporter plasmid.
HPV16 L1 and L2 coding plasmid along with pEGFP-N1 were amplified in E. coli host and were extracted before transfection with DNA-calcium phosphate deposition method into the HEK 293FT cells. The expression of HPV16 pseudoviruses was confirmed by fluorescence microscopy and flow cytometry. Pseudoviruses were partially purified with gel chromatography and then were visualized by atomic force microscopy.
Simultaneous transfection of HPV16 L1 and L2 coding plasmid along with pEGFP-N1 into the HEK 293FT cells resulted in self-assembly of 45-55 nm pseudoviruses, harboring the reporter gene coding plasmid.
In this project, HPV16 pseudoviruses, harboring a reporter gene were produced and partially purified, and their infecting capability was assessed. The pseudovirions could be used for neutralization assay and also as vectors for gene therapy.

Extracellular vesicles (EVs) contain active biological compounds which play important roles in biological processes. The secretion of EVs is a common phenomenon which occurs in archaea, bacteria and mammalian cells. The secretion of bacterial EVs has been discovered in various species of both Gram-negative and Gram-positive bacteria. Faecalibacterium prausnitzii is one of the commensal bacteria in human intestinal tract which has potentially therapeutic effects by secretion of bioactive compounds. Within the last few years, many investigations have been performed with respect to extracting and obtaining EVs and as a result, there are many methods to isolate and characterize EVs. The aim of this study was to isolate EVs from F. prausnitzii strain A2-165 and to characterize their physico-chemical properties.
EVs were isolated from F. prausnitzii strain A2-165 using ultracentrifugation and filtration. The EVs of bacterium were then characterized by Scanning Electron Microscopy, SDS-PAGE, Bradford assay, NanoDrop and Limulus Amebocyte Lysate (LAL) test.
The extracted EVs were confirmed with the shape of vesicles and the sizes ranging from ~ 30 to 250 nm. Total protein concentration of EVs were ~ 3 mg/ml from two methods; Bradford and NanoDrop, respectively. Protein profile pattern of F. prausnitzii-derived EVs ranged from 11 up to 245 kDa. Endotoxin measurement was 2.04 EU/ml.
The results of the current study demonstrated that EVs purity and conformation were acceptable. However, further investigations are necessary to elucidate the safety, efficacy, practicality and mechanism of action of this bacterium EVs in clinical practices, especially as vaccine delivery vehicles in the field of vaccine research.

Dendritic cells (DCs) have been recently employed as carriers for vaccines against several viral infections. The present study was designed to develop a prophylactic vaccine against hepatitis C virus (HCV) using DCs treated with Berberis vulgaris root extract (BRE), as a preclinical study.
BRE was prepared and injected to female BALB/c mice for DCs expansion. The expanded splenocyte cells (EDC) were purified and efficiently loaded ex vivo with HCV-nonstructural protein 3, NS3, (EDC-NS3). Mice were subcutaneously inoculated with EDC-NS3 vaccine candidate thrice with 4-week-intervals and IL-12, IFN-γ, IL-4, IL-10, MHC class II, CD3, CD16, indoleamine 2, 3 dioxygenase (IDO) and total protein levels were measured, post-vaccination by PCR and flow cytometry. Moreover, cytotoxic T lymphocyte and humoral immune responses were examined.
Our data revealed that immunization with EDC-NS3 vaccine elevated IL-12, IFN-γ and IL-4 expressions as well as MHC II and CD16 at protein levels. It also elicited strong HCV-NS3-specific humoral and cellular immune responses. However, the expressions of CD3, IDO, and IL-10 were down-regulated, post-vaccination.
EDC-NS3 immunization serves as an innovative modality for immunoprophylaxis against HCV infection.

Acinetobacter baumannii as a Gram-negative coccobacillus has become a major cause of hospital-acquired infections. The virulence factors involved in serum resistance are important targets in the development of an effective vaccine against this pathogen. Our aim in this project was in silico analyses of A. baumannii proteins involved in serum resistance which could potentially be used as efficient vaccines.
Based on computational procedures, we evaluated all A. baumannii proteins involved in serum resistance, namely AbOmpA, PKF, PLD , PBP 7/8, CipA and Tuf SurA1, as vaccine candidates. Subcellular localization, sequence conservation, domain prediction and 3D modelings were analyzed by online tools. Moreover, the prevalence of serum resistance factors in 5 strains of A. baumannii was characterized. The MHC-binding sites of class I and II were detected. Linear and conformational B cell epitopes were analyzed by 2 prediction servers.
The MetaLocGramN server showed that AbOmpA, PKF, PBP7/8, phospholipase D, CipA, Tuf and SurA1 were outer membrane protein (56.32%), extracellular protein (58.74%), extracellular protein (52.59%), cytoplasmic protein (45.08%), extracellular protein (53.8%), Cytoplasmic protein (96.36%) and extracellular protein (58.23%), respectively. The OMD of AbOmpA, PKF, PBP7/8 and phospholipase D, CipA, Tuf and SurA1 were 0.060, 0.076, 0.08, 0.101, 0.09, 0.06 and 0.103, respectively. The numbers of immunogenic linear and conformational epitopes with high score (P ≥ 0.6), extracted from beta-barrel of AbOmpA were 6 and 4; whereas these values for PKF were 10 and 4, respectively.
The in silico analyses and reverse vaccinology criteria showed that AbOmpA and PKF had better attributes as vaccine targets and they could be considered as promising vaccine candidates against A. baumannii.

Transient expression is an efficient and fast system to express recombinant proteins which has been used in different eukaryotic hosts such as mammalian and plant cells. Several applications of this system have so far been used which expression of proteins of interest is one of them. Recently, plants have attracted attention for being used as hosts for the production of recombinant pharmaceutical proteins such as antibodies and vaccines due to their lower price of production, compared to the mammalian systems. Many studies have conducted on the rapid production of vaccine candidate proteins either as a monomer or virus-like particles during epidemics of fast-spreading diseases such as influenza. Virus-like particles have been expressed in prokaryotic and eukaryotic systems are demonstrated to be one of the best antigen presenting systems which can carry antigens in a safe repetitive format on a particle to induce immunity.. Here, we present recent advances in applying transient expression in plants that can be used to produce naked or enveloped virus-like particles as vaccine candidate as well some clinical trial studies.

Hepatitis E virus (HEV) is a fecal-oral transmitting virus which causes a chronic liver disease. ORF2 is an immunogen capsid protein of HEV that has been proposed to be used for Hepatitis E vaccine design. It is a 660-amino acid protein which includes an immunogenic region (residues 112-607). This protein has been expressed in complete and truncated forms, using different expression vectors such as pRSET-C, pMAL, pSG and baculovirus expression systems. Escherichia coli BL21 which is used as a host for protein expression was utilized as a host for pET26b vector in this study. We evaluated the expression of ORF2 as Hepatitis E vaccine candidate in presence of several IPTG concentrations.
First, orf2 gene was sub-cloned into a pET26b vector which adds a C-terminal His-tag to the coding sequence. The procedure was confirmed by gel electrophoresis and double digestion. Subsequently, the recombinant pet26b-ORF2 was transformed into E. coli BL21 cells for protein expression and the resulted recombinant protein was analyzed by Bradford assay, SDS-PAGE and Western blotting.
SDS-PAGE and Western blotting confirmed the proper protein expression while there was no significant difference among the expressions of protein in presence of different IPTG concentrations.
The expression of HEV ORF2 protein was successfully performed in E. coli BL21 and it showed that ORF2 can be expressed in presence of different concentration of IPTG with no significant difference in protein expression. The produced recombinant protein could be used in further vaccine-related studies and also its expression can be studied at several different temperatures.