ABSTRACT
Neospora caninum is an apicomplexan protozoan that causes neosporosis, which has a high economic impact on cattle herds with no available vaccine. During infection, the secretion of dense granules and the expression of surface antigens play an important role in hosting immunomodulation. However, some epitopes of those antigens are immunogenic, and using these fractions could improve the subunit antigens in vaccine design. This study evaluates the recombinant peptides rsNcGRA1 and rsNcSAG4 derived from NcGRA1 and NcSAG4 native antigens as vaccine candidates produced by a fermentative process in the yeast culture system of Komagataella phaffii strain Km71, confirmed by colony PCR, SDS-PAGE, and western blotting. The assay was conducted in BALB/c mice using the peptides at low (25 µg) and standard (50 µg) dosages in monovalent and combined administrations at three time points with saponin as an adjuvant assessing the immunogenicity by antibodies response and cytokine production. We challenge the females after pregnancy confirmation using 2 × 105 NC-1 tachyzoites previously propagated in Vero cells. We assessed the chronic infection in dams and vertical transmission in the offspring by PCR and histopathology. Mice, especially those immunised with combined peptides and monovalent rsNcGRA1 at a standard dose, controlling the chronic infection in dams with the absence of clinical manifestations, showed an immune response with induction of IgG1, a proper balance between Th1/Th2 cytokines and reduced vertical transmission in the pups. In contrast, dams inoculated with a placebo vaccine showed clinical signs, low-scored brain lesions, augmented chronic infection with 80% positivity, 31% mortality in pups, and 81% vertical transmission. These findings indicate that rsNcGRA1 peptides in monovalent and combined with rsNCSAG4 at standard dose are potential vaccine candidates and improve the protective immune response against neosporosis in mice.
Subject(s)
Coccidiosis , Neospora , Protozoan Vaccines , Animals , Female , Mice , Pregnancy , Antibodies, Protozoan , Antigens, Protozoan , Chlorocebus aethiops , Coccidiosis/veterinary , Cytokines , Epitopes , Immunity , Infectious Disease Transmission, Vertical/prevention & control , Mice, Inbred BALB C , Neospora/genetics , Persistent Infection , Vaccination , Vero CellsABSTRACT
BACKGROUND: The immune system is able to recognize substances that originate from inside or outside the body and are potentially harmful. Foreign substances that bind to immune system components exhibit antigenicity and are defined as antigens. The antigens exhibiting immunogenicity can induce innate or adaptive immune responses and give rise to humoral or cell-mediated immunity. The antigens exhibiting mitogenicity can cross-link cell membrane receptors on B and T lymphocytes leading to cell proliferation. All antigens vary greatly in physicochemical features such as biochemical nature, structural complexity, molecular size, foreignness, solubility, and so on. OBJECTIVE: Thus, this review aims to describe the molecular bases of protein-antigenicity and those molecular bases that lead to an immune response, lymphocyte proliferation, or unresponsiveness. CONCLUSION: The epitopes of an antigen are located in surface areas; they are about 880-3,300 Da in size. They are protein, carbohydrate, or lipid in nature. Soluble antigens are smaller than 1 nm and are endocytosed less efficiently than particulate antigens. The more the structural complexity of an antigen increases, the more the antigenicity increases due to the number and variety of epitopes. The smallest immunogens are about 4,000-10,000 Da in size. The more phylogenetically distant immunogens are from the immunogen-recipient, the more immunogenicity increases. Antigens that are immunogens can trigger an innate or adaptive immune response. The innate response is induced by antigens that are pathogen-associated molecular patterns. Exogenous antigens, T Dependent or T Independent, induce humoral immunogenicity. TD protein-antigens require two epitopes, one sequential and one conformational to induce antibodies, whereas, TI non-protein-antigens require only one conformational epitope to induce low-affinity antibodies. Endogenous protein antigens require only one sequential epitope to induce cell-mediated immunogenicity.
Subject(s)
Carrier Proteins , T-Lymphocytes , Epitopes , Cell MembraneABSTRACT
This study investigated protection against Eimeria tenella following the vaccination of chicks with 5.3 × 106 E. tenella whole-sporozoites emulsified in the nanoparticle adjuvant IMS 1313 N VG Montanide™ (EtSz-IMS1313). One-day-old specific pathogen-free (SPF) chicks were subcutaneously injected in the neck with EtSz-IMS1313 on the 1st and 10th days of age. Acquired immunity was assayed through a challenge with 3 × 104 homologous sporulated oocysts at 21 days of age. The anticoccidial index (ACI) calculated for every group showed the effectiveness of EtSz-IMS1313 as a vaccine with an ACI of 186; the mock-injected control showed an ACI of 18 and the unimmunized, challenged control showed an ACI of -28. In a comparison assay, antibodies from rabbits and SPF birds immunized with EtSz-IMS1313 recognized almost the same polypeptides in the blotting of E. tenella sporozoites and merozoites. However, rabbit antisera showed the clearest recognition pattern. Polypeptides of 120, 105, 94, 70, 38, and 19 kDa from both E. tenella life cycle stages were the most strongly recognized by both animal species. The E. tenella zoite-specific IgG antibodies from the rabbits demonstrated the feasibility for successful B cell antigen identification.
ABSTRACT
Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.
Subject(s)
Crotalid Venoms , Animals , Antivenins/pharmacology , Crotalus , Mexico , Mice , Neutralization Tests , Rabbits , Recombinant Fusion ProteinsABSTRACT
Snakebite in Mexico is commonly treated with an antivenom which uses Bothrops asper and Crotalus simus venoms as immunogens. Current taxonomic recommendations for the C. simus species complex suggest a novel endemic species from Mexico: Crotalus mictlantecuhtli. The aim of this report was to evaluate the immunogenic properties of C. mictlantecuhtli venom and its potential to generate polyclonal antibodies capable of neutralizing other pitviper venoms. We generated an experimental anti-Crotalus mictlantecuhtli serum, using the rabbit model, to test recognition and neutralizing capacity against the homologous venom as well as venoms from C. atrox, C.basiliscus, C. durissus terrificus, C. scutulatus salvini, C. tzabcan and Ophryacus sphenophrys. Pre-incubation neutralization experiments using our experimental serum showed positive results against venoms containing crotoxin, while venoms from two non-neurotoxic pit-vipers were not neutralized. Rescue experiments in mice showed that, when intravenously injected (i.v.), C. mictlantecuhtli venom is not neutralized by a maximum dose of Antivipmyn® and the experimental serum after 5 min of envenomation, albeit mice envenomated intraperitoneally (i.p.) and rescued i.v. with Antivipmyn® survived even at 50 min after envenomation. Our results highlight the importance of using the highly neurotoxic C. mictlantecuhtli venom to increase antivenom effectiveness against Mexican neurotoxic pitvipers.
Subject(s)
Antivenins , Crotalus , Animals , Antibody Formation , Crotalid Venoms , Crotoxin , Mexico , Rabbits , Snake BitesABSTRACT
Chagas disease is an endemic chronic parasitosis in Latin America affecting more than 7 million people. Around 100 million people are currently at risk of acquiring the infection; however, no effective vaccine has been developed yet. Trypanosoma cruzi is the etiological agent of this parasitosis and as an intracellular protozoan it can reside within different tissues, mainly muscle cells, evading host immunity and allowing progression towards the chronic stage of the disease. Considering this intracellular parasitism triggers strong cellular immunity that, besides being necessary to limit infection, is not sufficient to eradicate the parasite from tissues, a differential immune response is required and new strategies for vaccines against Chagas disease need to be explored. In this work, we designed, cloned and expressed a chimeric molecule, named NCz-SEGN24A, comprising a parasite antigen, the N-terminal domain of the major cysteine protease of T. cruzi, cruzipain (Nt-Cz), and a non-toxic form of the staphylococcal superantigen (SAg) G, SEG, with the residue Asn24 mutated to Ala (N24A). The mutant SAg SEGN24A, retains its ability to trigger classical activation of macrophages without inducing T cell apoptosis. To evaluate, as a proof of concept, the immunogenicity and efficacy of the chimeric immunogen vs. its individual antigens, C3H mice were immunized intramuscularly with NCz-SEGN24A co-adjuvanted with CpG-ODN, or the recombinant proteins Nt-Cz plus SEGN24A with the same adjuvant. Vaccinated mice significantly produced Nt-Cz-specific IgG titers after immunization and developed higher IgG2a than IgG1 titers. Specific cell-mediated immunity was assessed by in-vivo DTH and significant responses were obtained. To assess protection, mice were challenged with trypomastigotes of T. cruzi. Both schemes reduced the parasite load throughout the acute phase, but only mice immunized with NCz-SEGN24A showed significant differences against control; moreover, these mice maintained 100% survival. These results encourage testing mutated superantigens fused to specific antigens as immune modulators against pathogens.
Subject(s)
Antigens, Bacterial/immunology , Chagas Disease/prevention & control , Cross Protection/immunology , Cysteine Endopeptidases/immunology , Protozoan Proteins/immunology , Superantigens/immunology , Trypanosoma cruzi/immunology , Animals , Antibodies, Neutralizing , Antibodies, Protozoan/immunology , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Antigens, Protozoan/immunology , Chagas Disease/immunology , Chagas Disease/parasitology , Cysteine Endopeptidases/genetics , Disease Models, Animal , Immunity, Cellular , Immunity, Humoral , Immunization , Mice , Parasite Load , Protein Conformation , Protein Domains/immunology , Protozoan Proteins/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Superantigens/chemistry , Superantigens/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Scorpion and spider envenomation is treated with the appropriate antivenoms, prepared as described by Césaire Auguste Phisalix and Albert Calmette in 1894. Such treatment requires the acquisition and manipulation of arachnid venoms, both very complicated procedures. Most of the toxins in the venoms of spiders and scorpions are extremely stable cysteine-rich peptide neurotoxins. Many strategies have been developed to obtain synthetic immunogens to facilitate the production of antivenoms against these toxins. For example, whole peptide toxins can be synthesized by solid-phase peptide synthesis (SPPS). Also, epitopes of the toxins can be identified and after the chemical synthesis of these peptide epitopes by SPPS, they can be coupled to protein carriers to develop efficient immunogens. Moreover, multiple antigenic peptides with a polylysine core can be designed and synthesized. This review focuses on the strategies developed to obtain synthetic immunogens for the production of antivenoms against the toxic Cys-rich peptides of scorpions and spiders.
ABSTRACT
BACKGROUND: Pathogens use multiple mechanisms to disrupt cell functioning in their host and allow pathogenesis. These mechanisms involve communication between the pathogen and the host cell through protein-protein interactions. METHODS: Protein-protein interactions chains referred to as signal transduction pathways are the processes by which a chemical or physical signal transmits through a cell as series of molecular events so the pathogen needs to intercept these molecular pathways at few positions to induce pathogenesis such as pathogen viability, infection or hypersensitivity. RESULTS: The pathogen nodes of interception are not necessarily the most immunogenic; so that novel immunogenicity-improvement strategies need to be developed thought a chemical conjugation of the pathogen-carrier nodes to develop an efficient immune response in order to block pathogenesis. On the other hand, if pathogen-carriers are immunogens; toleration ought to be induced by this conjugation avoiding hypersensitivity. Thus, this paper addresses the biological plausibility of plant-phenolics as pathogen-carrier immunogenicity modulator haptens. CONCLUSION: The plant-phenolic compounds have in their structure functional groups such as hydroxyl, carbonyl, carboxyl, ester, or ether, capable of reacting with the amino or carbonyl groups of the amino acids of a pathogen-carrier to form conjugates. Besides, the varied carbon structures these phenolic compounds have; it is possible to alter the pathogen-carrier related factors that determine the immunogenicity: 1) Structural complexity, 2) Molecular size, 3) Structural heterogeneity, 4) Accessibility to antigenic determinants or epitopes, 5) Optical configuration, 6) Physical state, or 7) Molecular rigidity.
Subject(s)
Adaptive Immunity/drug effects , Haptens/immunology , Host-Pathogen Interactions/immunology , Immunity, Innate/drug effects , Phenols/immunology , Plants/immunology , Adaptive Immunity/immunology , Amino Acids/chemistry , Amino Acids/immunology , Animals , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Humans , Immunity, Innate/immunology , Phenols/chemistry , Plants/chemistry , Signal TransductionABSTRACT
We describe the intestinal changes and biological parameters of the tick species Rhipicephalus microplus exposed to the immune response of calves vaccinated with two subunits of immunogens. The first group of Bos taurus calves was immunized with a synthetic peptide (SBm7462), whereas the second group received an inoculum for synthetic control. The third group was immunized with a recombinant peptide (rSBm7462); an inoculum was injected into a fourth group of calves for recombinant control. Each formulation was administered to these calves during three times at intervals of 30 days. At 21 days after the last immunization, the calves were challenged using a total of 4500 larvae per animal. Indirect ELISA was realized to identify the kinetics of IgGs from samples of calves studied. Naturally detaching ticks were collected for analyses of biological performance and histological changes in the midgut. We dissected randomly detached ticks. The midgut of each of these ticks was removed and processed routinely for histology, stained with hematoxylin-eosin (H&E) and slow Giemsa. Slides were also subjected to immunohistochemistry. The antibody response showed significant induction of high-affinity IgGs in calves immunized with both peptides in comparison to calves of the control groups. Histological changes included damage of the intestinal epithelium in ticks fed on immunized hosts and intense immunostaining in midgut cells, using the serum of calves immunized with recombinant peptide. There were significant differences in all biological performing parameters of ticks detached from vaccinated calves in comparison with ticks of the control groups. We identified reductions of 87.7 and 93.5% in engorged ticks detached from calves immunized with a synthetic and recombinant peptides, respectively, a 28 and 8.60% lower egg mass in groups immunized with synthetic and recombinant peptides, respectively, and a 38.4% reduction of the value of nutrient index/tick in the group immunized with the recombinant peptide. Our findings show that the immune response induced by small peptides in cattle can modify the digestion and metabolism of ticks fed on vaccinated animals, resulting in changes in tick performance.
Subject(s)
Antigens/therapeutic use , Cattle Diseases/parasitology , Cattle/parasitology , Rhipicephalus/pathogenicity , Tick Infestations , Vaccines/therapeutic use , Animals , Immunization , IntestinesABSTRACT
Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen's formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.
Subject(s)
Antibodies, Neutralizing/pharmacology , Antivenins/pharmacology , Phosphoric Diester Hydrolases/toxicity , Spider Venoms/toxicity , Animals , Antivenins/metabolism , Edema/chemically induced , Edema/drug therapy , Humans , Male , Metalloproteases/metabolism , Mice, Inbred BALB C , Necrosis/chemically induced , Necrosis/drug therapy , Phosphoric Diester Hydrolases/metabolism , Platelet Aggregation/drug effects , Rabbits , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Spider Venoms/metabolism , SpidersABSTRACT
Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.
Acidentes em humanos com aranhas do gênero Loxosceles são um importante problema de saúde afetando milhares de pessoas em todo o mundo. Os pacientes evoluem para lesões locais graves e, em muitos casos, para distúrbios sistêmicos como insuficiência renal aguda, casos em que os antivenenos são considerados tratamento mais eficaz. Entretanto, para a produção de antiveneno, a extração do veneno utilizado no processo de imunização é trabalhosa e o rendimento é muito baixo. Assim, muitos grupos têm explorado o uso de toxinas recombinantes de Loxosceles, particularmente fosfolipases D (FLDs), para produzir o antiveneno. No entanto, algumas atividades importantes do veneno não são neutralizadas pelos anticorpos anti-FLD. As metaloproteases do tipo astacinas (MPTAs) são a segunda toxinas mais expressas, agindo na matriz extracelular, indicando a importância de sua inclusão na formulação do antígeno para fornecer um melhor antiveneno. Aqui mostramos a construção de um imunógeno híbrido recombinante, chamado LgRec1ALP1, composto de regiões hidrofílicas das FLD e MPTA de Loxosceles gaucho. Embora o LgRec1ALP1 tenha sido expresso como corpúsculo de inclusão, resultou em bons rendimentos e foi eficaz para poduzir anticorpos neutralizantes em camundongos. O antissoro neutralizou atividades fibrinogenolítica, de agregação plaquetária e dermonecróticas induzidas pelos venenos de L. gaucho, L. laeta e L. intermedia, indicando que o antígeno híbrido recombinante pode ser uma fonte valiosa para a produção de anticorpos protetores contra os venenos de Loxosceles ssp. Além disso, a abordagem de toxina híbrida recombinante pode enriquecer e expandir alternativas de antígenos para produção de antissoros para outros venenos.
ABSTRACT
Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.
ABSTRACT
Zika virus (ZIKV) infection has extended rapidly all over the world in the last decades affecting humans of all ages, inducing severe illness such as the autoimmune Guillain-Barré syndrome as well as fetal neurodevelopmental defects. Despite the epidemiological importance of ZIKV, today there are no commercially available drugs or vaccines to combat or prevent this infection. Microalgae are attractive hosts to produce and deliver vaccines, with some candidates under preclinical evaluation. Herein, algae-based expression was assessed for the production of a new vaccine candidate against ZIKV called ZK. The Algevir technology was applied to express an antigenic protein called ZK comprising the B subunit of the heat labile Escherichia coli enterotoxin along with 3 epitopes from the ZIKV envelope glycoprotein. Efficient expression of the ZK antigen was achieved in Schizochytrium sp. with yields of up to 365⯵g g-1 microalgae fresh weight. Upon oral administration in mice, the microalgae-made ZK protein elicited significant humoral responses at a higher magnitude to those induced upon subcutaneous immunization. The algae-made ZK vaccine represents a promising candidate to formulate attractive vaccines against ZIKV.
Subject(s)
Antigens, Viral/genetics , Epitopes/genetics , Microalgae/genetics , Stramenopiles/genetics , Viral Envelope Proteins/genetics , Viral Vaccines , Zika Virus/genetics , Administration, Oral , Animals , Antigens, Viral/immunology , Epitopes/immunology , Female , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Mice, Inbred BALB C , Mucous Membrane/immunology , Viral Envelope Proteins/immunology , Zika Virus/immunology , Zika Virus Infection/prevention & controlABSTRACT
Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibody Specificity , Antigens/administration & dosage , Antigens/immunology , Immunization/methods , Immunodominant Epitopes/immunology , Animals , Animals, Newborn , Humans , Immune Tolerance , Immunization ScheduleABSTRACT
Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of PfCSP, STARP; MSA1 and Pf155/RESA from pre- and erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei-ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems.
Subject(s)
Antigens, Protozoan/immunology , Malaria/immunology , Malaria/prevention & control , Peptides/immunology , Animals , Epitopes/immunology , Malaria Vaccines/immunology , Malaria Vaccines/therapeutic use , Mice , Mice, Inbred BALB C , Plasmodium falciparum/immunology , Plasmodium falciparum/pathogenicity , Protozoan Proteins/immunologyABSTRACT
This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD50) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 µL of serum from any of the immunized rabbits were enough to neutralize three LD50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications.
Subject(s)
Antivenins/chemistry , Recombinant Proteins/chemistry , Scorpion Venoms/chemistry , Scorpions/chemistry , Amino Acid Sequence , Animals , Antivenins/genetics , Antivenins/pharmacology , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Female , Immunization , Lethal Dose 50 , Mexico , Mice , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Scorpion Venoms/genetics , Scorpion Venoms/pharmacology , Sequence AlignmentABSTRACT
A Pseudomonas aeruginosa é agente etiológico de infecções oportunistas, principalmente em pacientes imunocomprometidos. Suas características inerentes em desenvolver resistência aos mais variados tipos de antibacterianos a torna um ponto crítico no controle de infecções. Em animais, os problemas com multirresistência ocorrem principalmente em casos de otite, cistite, úveo-conjuntivite, endometrite e mastite, não havendo vacina comercialmente disponível. No intuito de melhorar a imunogenicidade desse antígeno, foi testada a técnica de conjugação do lipopolissacarídeo (LPS) de P. aeruginosa à albumina bovina (BSA) por aminação redutiva direta utilizando .-periodato de sódio. A conjugação foi avaliada por cromatografia de gel-permeação, dosando-se açúcar e proteína totais, e tanto o LPS quanto a BSA foram identificados em proporções semelhantes. A imunização de camundongos com a vacina conjugada LPS-BSA conferiu títulos de anticorpos aglutinantes contra P. aeruginosa inferiores aos obtidos com a mistura de LPS e BSA livres. Foram 65% e 86% menores na 6ª e na 10ª semanas após o procedimento de hiperimunização, respectivamente. Isto indica que a reação de conjugação resultou em um produto imunogênico, porém, sua qualidade precisará ser melhorada.
Pseudomonas aeruginosa is an etiologic agent of opportunistic infections mainly in immunocompromised patients. Its inherent feature of developing resistance to a wide range of antibacterial agents make it a critical point in infection control. In animals, problems with multidrug resistance occur in otitis, cystitis, uveo-conjunctivitis, endometritis and mastitis, and there is no commercially available vaccine. With the aim of improving its immunogenicity, the lipopolysaccharide (LPS) antigen was coupled to bovine serum albumin (BSA) with .-periodate as the reductive agent. The conjugation was evaluated by gel-permeation chromatography, by quantitating total sugar and protein, and both LPS and BSA were detected in similar proportions. The immunization of mice with LPS-BSA conjugate vaccine resulted in an agglutinating antibody response against P. aeruginosa lower than that obtained for a mixture of free LPS and BSA. They were 65% and 86% lower in the 6th and 10th weeks after the hyperimmunization procedure, respectively. This indicates that the conjugation reaction resulted in an immunogenic product, however its quality should be improved.
Subject(s)
Animals , Cattle , Pseudomonas aeruginosa , Serum Albumin, Bovine , Cattle Diseases , LipopolysaccharidesABSTRACT
ABSTRACT Pseudomonas aeruginosa is an etiologic agent of opportunistic infections mainly in immunocompromised patients. Its inherent feature of developing resistance to a wide range of antibacterial agents make it a critical point in infection control. In animals, problems with multidrug resistance occur in otitis, cystitis, uveo-conjunctivitis, endometritis and mastitis, and there is no commercially available vaccine. With the aim of improving its immunogenicity, the lipopolysaccharide (LPS) antigen was coupled to bovine serum albumin (BSA) with .-periodate as the reductive agent. The conjugation was evaluated by gel-permeation chromatography, by quantitating total sugar and protein, and both LPS and BSA were detected in similar proportions. The immunization of mice with LPS-BSA conjugate vaccine resulted in an agglutinating antibody response against P. aeruginosa lower than that obtained for a mixture of free LPS and BSA. They were 65% and 86% lower in the 6th and 10th weeks after the hyperimmunization procedure, respectively. This indicates that the conjugation reaction resulted in an immunogenic product, however its quality should be improved.
RESUMO A Pseudomonas aeruginosa é agente etiológico de infecções oportunistas, principalmente em pacientes imunocomprometidos. Suas características inerentes em desenvolver resistência aos mais variados tipos de antibacterianos a torna um ponto crítico no controle de infecções. Em animais, os problemas com multirresistência ocorrem principalmente em casos de otite, cistite, úveo-conjuntivite, endometrite e mastite, não havendo vacina comercialmente disponível. No intuito de melhorar a imunogenicidade desse antígeno, foi testada a técnica de conjugação do lipopolissacarídeo (LPS) de P. aeruginosa à albumina bovina (BSA) por aminação redutiva direta utilizando .-periodato de sódio. A conjugação foi avaliada por cromatografia de gel-permeação, dosando-se açúcar e proteína totais, e tanto o LPS quanto a BSA foram identificados em proporções semelhantes. A imunização de camundongos com a vacina conjugada LPS-BSA conferiu títulos de anticorpos aglutinantes contra P. aeruginosa inferiores aos obtidos com a mistura de LPS e BSA livres. Foram 65% e 86% menores na 6ª e na 10ª semanas após o procedimento de hiperimunização, respectivamente. Isto indica que a reação de conjugação resultou em um produto imunogênico, porém, sua qualidade precisará ser melhorada.