Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.
Tuberculosis (TB) is one of the deadliest human diseases worldwide caused by mycobacterial infection in the lung. Bacillus Calmette-Guerin (BCG) vaccine protects against disseminated TB in children, but its effectiveness is still questionable due to highly variable protections in adolescence and elderly individuals. Targeting the latency M.tb antigen is a recent therapeutic approach to eradicate dormant pathogen that could possibly lead to disease activation. In this study, we aimed to potentiate immune responses elicited against 16 kDa α-crystalline (HspX) tuberculosis latency antigen by incorporation of Combined Adjuvant for Synergistic Activation of Cellular immunity (CASAC). Histidine-tagged recombinant HspX protein was initially produced in Escherichia coli and purified using Ni-NTA chromatography. To evaluate its adjuvanticity, C57BL/6 mice (n = 5) were initially primed and intradermally immunised in 2-weeks interval for 4 rounds with recombinant HspX, formulated with and without CASAC. Humoral and cell-mediated immune responses elicited against HspX antigen were evaluated using ELISA and Flow Cytometry. Our findings showed that CASAC improved humoral immunity with increased antigen-specific IgG1 and IgG2a antibody response. Stronger CD8+ and Th1-driven immunity was induced by CASAC formulation as supported by elevated level of IFN-γ, TNF-α, IL-12 and IL-17A; and with low IL-10 secretion. Interestingly, adjuvanted HspX vaccine triggered a higher percentage of effector memory T-cell population than those immunised with unadjuvanted vaccine. In conclusion, CASAC adjuvant has great potential to enhance immunogenicity elicited against HspX antigen, which could be an alternative regimen to improve the efficacy of future therapeutic vaccine against Mycobacterium tuberculosis.
Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.
Development of vaccines for infectious diseases has come a long way with recent advancements in adjuvant developments and discovery of new antigens that are capable of eliciting strong immunological responses for sterile eradication of disease. Tuberculosis (TB) that kills nearly 2 million of the population every year is also one of the highlights of the recent developments. The availability or not of diagnostic methods for infection has implications for the control of the disease by the health systems but is not related to the immune surveillance, a phenomenon derived from the interaction between the bacteria and their host. Here, we will review the immunology of TB and current vaccine candidates for TB. Current strategies of developing new vaccines against TB will also be reviewed in order to further discuss new insights into immunotherapeutic approaches involving adjuvant and antigens combinations that might be of potential for the control of TB.
Lymphatic filariasis is a neglected parasitic disease that affects millions in tropical and subtropical countries and is caused by Wuchereria and Brugia species. Specific and sensitive detection methods are essential in mapping infected areas where rapid tests are needed to cover underdeveloped and remote regions, which facilitates eliminating the disease as a public health problem. A few commercialized rapid tests based on antigen or antibody detection are available, but the former only detects infection by Wuchereria species and cross-reacts with nonlymphatic filaria, whereas antibody detection might provide positive results of previous infection. Here, we report the production of three different recombinant immunoglobulin gamma (IgG)1 antibodies based on scFvs previously generated via human antibody phage display technology, that is, anti-BmR1 clone 4, anti-BmXSP clone 5B, and anti-BmXSP clone 2H2. The scFv sequences were cloned into a pCMV-IgG1 vector, then transfected into a HEK293F cell line. The generated antibodies were found to be able to bind to their respective targets even at relatively low concentration. Conjugation of Fc to scFv induces binder stability and provides multiple labeling sites for probes and signaling molecules that can be used in rapid tests.