Biotechnology

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Designing T-cell epitope-based vaccine against Eimeria infection in chicken using immunoinformatics approach.
(2021) Madlala, Thabile.; Adeleke, Matthew Adekunle.; Okpeku, Moses.
Chicken coccidiosis is the most significant ubiquitous, intestinal parasitic disease known to infect chickens globally. It is recognised for incurring significant production loss to the poultry industry, caused by single or multiple Eimeria spp. infections which threaten chicken welfare and productivity. The emergence of drug resistance in parasites and pathogenicity reversion has put pressure on the poultry industry to reduce chemoprophylactic drugs and live vaccines as preventive measures against coccidiosis. Recombinant DNA vaccines have shown promising results as an alternative option, but complete protection has not been reported highlighting the need for the design of new vaccine against this disease. In this study, Eimeria antigens Immune Mapped Protein-1(IMP1) and Microneme Protein-2(MIC2) were explored using reverse vaccinology and immunoinformatics tool to predict and design potential multiepitope vaccine candidate against coccidiosis. A total of 28 and 19 antigenic T-cell epitopes were predicted and used to construct two multiepitope vaccines with 610 and 512 amino acids for IMP1 and MIC2, respectively. The produced vaccines exhibited favoured characteristics for an ideal vaccine candidate; they were antigenic (Vaxijen score of 0.5989 and 0.5103), immunogenic (scores: 10.15 and 9 419), thermostable (instability index <40 ), and non-allergic. The presence of IFN-gamma and IL-4 inducing epitopes in the constructed vaccine enables vaccine to trigger a cellular and humoral response within the host. Molecular docking of designed vaccines with toll-like receptors (TLR4 and TLR5) to determine vaccine interaction and stability was confirmed by molecular dynamics simulation root-mean square deviation (RMDS) and root-mean-square fluctuation (RMSF) analysis. The designed vaccines induced immune response through production of cytokines and antibodies associated with tertiary response. When exposed to online immune simulation C-ImmSim, both vaccines produced potent immune response through production of IgG, Tc and Th cell and memory Bcells. The constructed multiepitope vaccine in this present study is highly promising and as such further experimental work should be done to confirm its suitability against chicken coccidiosis.
Diversity of Eimeria tenella apical membrane antigen-1 from chickens in Mpumalanga province and its in silico epitope prediction as a vaccine candidate.
(2021) Tenza, Petronella Nokukhanya.; Adeleke, Matthew Adekunle.; Fatoba, Abiodun Joseph.
Coccidiosis has been a significant challenge in the poultry industry. There is a high request for the modification of a cost-effective immunizing agent to curtail this disease. Apical membrane antigen 1 (AMA1) has been reported as a protective antigen in sub-unit vaccine development against several apicomplexan parasites such as Plasmodium falciparum, Eimeria tenella and Eimeria maxima. However, knowledge of genetic diversity in this vaccine candidate is imperative. Also, to minimize the cost and time involved in producing a vaccine, computational vaccine design has received much attention through the immunoinformatics method. Therefore, screening for the potential vaccine epitopes in AMA1 that can induce cellular and humoral immune response through the immunoinformatics technique looks promising. This investigation aimed to detect the level of genetic diversity amid Eimeria tenella Apical Membrane Antigen 1(EtAMA1) in selected farms in Mpumalanga province and predict vaccine epitopes from this antigen. Four hundred fresh faecal samples were collected from 10 selected broiler chicken farms in Mpumalanga. The samples were screened for Eimeria oocyst using a compound microscope, and samples containing oocyst were further screened for E. tenella using molecular methods. AMA1 (n=103) was amplified from positive samples for E. tenella, and resulted amplicons were sent to Inqaba Biotec for sequencing and analyzed using MEGA6.06 and DnaSP programs. The results revealed low levels of genetic diversity among Mpumalanga EtAMA1 sequences which were measured by nucleotide diversity (0.0007) diversity, haplotype diversity (0.113) and haplotype number (3). Correspondingly, the haplotype network revealed 4 haplotypes, 3 of which consist of samples from Mpumalanga. Identification of immunogenic B- and T-cell epitopes from EtAMA1 sequences was further carried out and were used to construct a multiepitope vaccine (MEV) using immunoinformatics approaches. The constructed MEV is 311 amino acids long. It was constructed by linking 6 B-cell, 3 CD8+ epitopes and 6 CD4+ epitopes with appropriate adjuvant and linkers. Both adjuvant and linkers were used to increase the immunogenicity of the MEV. The designed MEV was highly antigenic and non-allergenic. The results showed a strong binding affinity of MEV with TLR4. These results suggest that the predicted vaccine could be a significant vaccine candidate against chicken coccidiosis through further experimental validation is still necessary.
Optimization of antifungal production by Bacillus species in the presence of nanoparticle supplementation and preliminary scale-up.
(2023) Nzimande, Sikhulile Nosimo.; Gueguim Kana, Evariste Bosco.; Ramchuran, Santosh Omrajah.; Sanusi, Adeyemi Isaac.
The intensive agricultural practices used to meet global crop production demands have resulted in the rigorous use of chemical pesticides. These ultimately compromise crop production as well as the environment. In order to alleviate this, cheaper and environmentally friendly, biocontrol agents have been considered as an alternative to chemical pesticides. Biosurfactants are a promising alternative to chemical pesticides due to their higher biodegradability, lower toxicity, and environmental friendliness. Amongst the many bacterial and fungal biosurfactant producers, biosurfactants from Bacillus species show promise as biocontrol agents. These biosurfactants are known for their wide biotechnological use in agricultural, industrial, and medicinal fields. However, large scale production is still faced with challenges such as low yields and high production cost thus raising the need for modelling, optimization, catalytic and scale up investigations. Hence, a study was undertaken with the aim of enhancing biosurfactant production through process modelling and optimization with subsequent assessment of the scale up potential of the optimized process. A Response Surface Methodology (RSM) using box Behnken design was used to investigate the optimal process conditions for improved biosurfactant production from B. subtilis BS20. The investigated process parameters included glucose concentration (10 – 30 g/L), incubation temperature (25 – 45℃) and incubation time (24 – 96 h). The developed model gave a high coefficient of determination (R2 ) = 0.86, p-value of 0.0279 and F-value of 4.62 for the modelled biosurfactant production. Optimized process conditions of 11.5 g/L glucose concentration, 24 h incubation time and 41o C for incubation temperature were obtained and produced a maximal antifungal activity of 68 mm. Moreover, supplementary inclusion of seven (7) different nanoparticles as a biocatalyst in the cultivation of B. subtilis BS20 was carried out using the optimal process condition to further improve antifungal (biosurfactant) production. The inclusion of nanoparticles favored increased biomass yield, but biosurfactant with high antifungal activity was not obtained. Moreover, when it comes to commercializing new bioprocess and bioproduct developments, bio – process scale-up in the biotechnology industry is an essential stage. This study therefore evaluated the scale up of biosurfactant production based on constant power consumption, Reynold number and impeller tip speed. The stirrer speed (n), impeller diameter (di), number of impellers (N), power number (Np), broth density (⍴), working volume and geometric factor (fc) were correlated with impeller tip speed (Vtip), Reynolds number (𝑅𝑅𝑅𝑅) and power consumption rate (P/V) to obtain the most suitable criterion for biosurfactant production in a 10 L bioreactor. Implementing constant Vtip value from the 1 L scale: 93 rpm, Reynold number (Re) 5.9E - 04, Power (P) 0.32 W, Power to Volume ratio (P/VL) 160 W/m3 , circulation time (tc) 5.2 s and shear stress (γ) 15.5 S-1, at 41 °C, gave the highest antifungal activity of 65 mm zone of inhibition in the 10 L scale bioreactor. The antifungal activity obtained for constant Vtip were comparable to those obtained at 1L bioreactors (57 mm), this showed that the bioprocess dynamics for achieving high antifungal activity are available, further paving the way for feasible commercialization strategies. This study has elucidated the optimum process conditions for B. subtilis BS20 metabolism for improved biosurfactant production resulting in significant antifungal activity. Furthermore, findings showed that the inclusion of nanoparticles biocatalyst to the process enhanced biomass yields. Process scale up provided preliminary data for large scale production of biosurfactant production from B. subtilis BS20.

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