Expression and preliminary characterization of the potential vaccine candidate LipL32 of leptospirosis
TL;DR: In this paper, the over expression of LipL32 and hGMCSF genes into yeast expression system for obtaining a high yield of recombinant protein production was investigated for leptospirosis.
Abstract: Leptospirosis is a globally re-emerging infectious disease mainly for mammals. The infection is caused by the spirochete Gram-negative bacterium Leptospira interrogans, which affects animals and humans worldwide. In our previous studies, recombinant protein production has been obtained from the bacterial expression system. In this study, we have investigated the over expression of LipL32 and hGMCSF genes into yeast expression system for obtaining a high yield of recombinant protein production. Here, we described the yeast expression studies with several applications such as protein folding, fast growth, and post-translational modification. The expression studies were carried out in a novel protein expression system, the methylotrophic yeast Pichia pastoris KM71 strain. The LipL32, Green fluorescent protein (EGFP), and human granulocyte–macrophage colony-stimulating factor (hGMCSF) genes were cloned into pPIC9 yeast expression vector. The recombinant clones of pPIC9-EGFP-LipL32 and pPIC9-EGFP-hGMCSF-LipL32 were transformed into Pichia pastoris KM71 strain by electroporation. Media optimization and other physiological characters were studied for the transformed recombinant protein. The protein was then purified using a Ni–NTA column; meanwhile, the recombinant DNA constructs contain His-tag at the C- terminal end. Finally, the intracellular EGFP expression of pPIC9-EGFP-LipL32, and pPIC9-EGFP-hGMCSF-LipL32 in Pichia pastoris KM71 strain was confirmed by fluorescence microscopic analysis. Protein–protein dockings were done to study LipL32-Adjuvant (hGMCSF, hIgGFC, and hC3d) interactions. Furthermore, this docking analysis was shown better interaction between LipL32, and hGMCSF, which is also used for the enhanced vaccine potential against leptospirosis.
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TL;DR: The experiments show that immunization with recombinant VM proteins prevents leptospirosis clinical pathogenesis and leads to markedly reduced key target organ infection in this animal model, and suggest the possibility that a VM protein-based, serovar-independent, pan-leptospIrosis vaccine may be feasible.
Abstract: The molecular and cellular pathogenesis of leptospirosis remains poorly understood. Based on comparative bacterial genomics data, we recently identified the hypothetical PF07598 gene family as encoding secreted exotoxins (VM proteins) that mediate cytotoxicity in vitro. To address whether VM proteins mediate in vivo leptospirosis pathogenesis, we tested the hypothesis that VM protein immunization of mice would protect against lethal challenge infection and reduce bacterial load in key target organs. C3H/HeJ mice were immunized with recombinant E. coli-produced, endotoxin-free, leptospiral VM proteins (derived from L. interrogans serovar Lai) in combination with the human-compatible adjuvant, glucopyranoside lipid A/squalene oil-in-water. Mice receiving full length recombinant VM proteins were protected from lethal challenge infection by L. interrogans serovar Canicola and had a 3-4 log10 reduction in bacterial load in the liver and kidney. These experiments show that immunization with recombinant VM proteins prevents leptospirosis clinical pathogenesis and leads to markedly reduced key target organ infection in this animal model. These data support the role of leptospiral VM proteins as virulence factors and suggest the possibility that a VM protein-based, serovar-independent, pan-leptospirosis vaccine may be feasible.
7 citations
TL;DR: The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products as mentioned in this paper . But despite these fast-developing approaches, subunit vaccines still show a wide spectrum of interesting potentialities and an important margin for further development.
Abstract: The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the COVID-19 pandemics have globally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiological needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80's, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products.
6 citations
TL;DR: In this paper , the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity.
Abstract: The first leptospiral recombinant vaccine was developed in the late 1990s. Since then, progress in the fields of reverse vaccinology (RV) and structural vaccinology (SV) has significantly improved the identification of novel surface-exposed and conserved vaccine targets. However, developing recombinant vaccines for leptospirosis faces various challenges, including selecting the ideal expression platform or delivery system, assessing immunogenicity, selecting adjuvants, establishing vaccine formulation, demonstrating protective efficacy against lethal disease in homologous challenge, achieving full renal clearance using experimental models, and reproducibility of protective efficacy against heterologous challenge. In this review, we highlight the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity.
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TL;DR: A complementary DNA for the Aequorea victoria green fluorescent protein produces a fluorescent product when expressed in prokaryotic or eukaryotic cells, which can be used to monitor gene expression and protein localization in living organisms.
Abstract: A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
7,016 citations
TL;DR: The completion of the genome sequence of Leptospira interrogans serovar lai, and other continuing leptospiral genome sequencing projects, promise to guide future work on the disease.
Abstract: In the past decade, leptospirosis has emerged as a globally important infectious disease. It occurs in urban environments of industrialised and developing countries, as well as in rural regions worldwide. Mortality remains significant, related both to delays in diagnosis due to lack of infrastructure and adequate clinical suspicion, and to other poorly understood reasons that may include inherent pathogenicity of some leptospiral strains or genetically determined host immunopathological responses. Pulmonary haemorrhage is recognised increasingly as a major, often lethal, manifestation of leptospirosis, the pathogenesis of which remains unclear. The completion of the genome sequence of Leptospira interrogans serovar lai, and other continuing leptospiral genome sequencing projects, promise to guide future work on the disease. Mainstays of treatment are still tetracyclines and beta-lactam/cephalosporins. No vaccine is available. Prevention is largely dependent on sanitation measures that may be difficult to implement, especially in developing countries.
2,055 citations
TL;DR: This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results of the ClusPro server.
Abstract: The ClusPro server (https://cluspro.org) is a widely used tool for protein-protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format. However, ClusPro also offers a number of advanced options to modify the search; these include the removal of unstructured protein regions, application of attraction or repulsion, accounting for pairwise distance restraints, construction of homo-multimers, consideration of small-angle X-ray scattering (SAXS) data, and location of heparin-binding sites. Six different energy functions can be used, depending on the type of protein. Docking with each energy parameter set results in ten models defined by centers of highly populated clusters of low-energy docked structures. This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results. Although the server is heavily used, runs are generally completed in <4 h.
1,699 citations
TL;DR: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins and the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed.
Abstract: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.
1,237 citations
TL;DR: Leptospirosis is among the leading zoonotic causes of morbidity worldwide and accounts for numbers of deaths, which approach or exceed those for other causes of haemorrhagic fever.
Abstract: Background
Leptospirosis, a spirochaetal zoonosis, occurs in diverse epidemiological settings and affects vulnerable populations, such as rural subsistence farmers and urban slum dwellers. Although leptospirosis is a life-threatening disease and recognized as an important cause of pulmonary haemorrhage syndrome, the lack of global estimates for morbidity and mortality has contributed to its neglected disease status.
Methodology/Principal Findings
We conducted a systematic review of published morbidity and mortality studies and databases to extract information on disease incidence and case fatality ratios. Linear regression and Monte Carlo modelling were used to obtain age and gender-adjusted estimates of disease morbidity for countries and Global Burden of Disease (GBD) and WHO regions. We estimated mortality using models that incorporated age and gender-adjusted disease morbidity and case fatality ratios. The review identified 80 studies on disease incidence from 34 countries that met quality criteria. In certain regions, such as Africa, few quality assured studies were identified. The regression model, which incorporated country-specific variables of population structure, life expectancy at birth, distance from the equator, tropical island, and urbanization, accounted for a significant proportion (R2 = 0.60) of the variation in observed disease incidence. We estimate that there were annually 1.03 million cases (95% CI 434,000–1,750,000) and 58,900 deaths (95% CI 23,800–95,900) due to leptospirosis worldwide. A large proportion of cases (48%, 95% CI 40–61%) and deaths (42%, 95% CI 34–53%) were estimated to occur in adult males with age of 20–49 years. Highest estimates of disease morbidity and mortality were observed in GBD regions of South and Southeast Asia, Oceania, Caribbean, Andean, Central, and Tropical Latin America, and East Sub-Saharan Africa.
Conclusions/Significance
Leptospirosis is among the leading zoonotic causes of morbidity worldwide and accounts for numbers of deaths, which approach or exceed those for other causes of haemorrhagic fever. Highest morbidity and mortality were estimated to occur in resource-poor countries, which include regions where the burden of leptospirosis has been underappreciated
1,090 citations