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INTRODUCTION: Producing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. OBJECTIVE: Considering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. METHODS: Subcutaneously, 18 Texel sheep received two doses (200 µg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). RESULTS: Both formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. CONCLUSION: While adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.
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Anticorpos Antibacterianos , Anticorpos Neutralizantes , Vacinas Bacterianas , Botulismo , Enterotoxemia , Animais , Botulismo/prevenção & controle , Botulismo/veterinária , Botulismo/imunologia , Ovinos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Anticorpos Antibacterianos/sangue , Enterotoxemia/prevenção & controle , Enterotoxemia/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Imunoglobulina G/sangue , Escherichia coli/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , FemininoRESUMO
Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is an important public health problem, occurring mainly in Latin America. The disease has a major social and economical effect, negatively impacting the life of the infected individuals, and bringing great costs to public health. An early and accurate diagnosis is essential for administration of early treatment. In addition, prognostic tests may aid disease management, decreasing hospitalization costs. However, the serological diagnostic scenario for CD still faces several challenges, making the development of new diagnostic kits a pressing matter. Facing this scenario, several researchers have expanded efforts in developing and testing new antigens, such as recombinant proteins and recombinant multiepitope proteins, with promising results. These recombinant antigens offer several advantages, such as improved sensitivity and specificity, in addition to facilitated scaling. Also, it has been possible to observe a rising number of studies using ELISA and point-of-care platforms, employing these antigens in the past few years. Among them, recombinant proteins were the most applied antigens, demonstrating great capacity to discriminate between positive and negative samples. Although fewer in number, recombinant multiepitope proteins also demonstrated an improved diagnostic performance. Indeed, a great number of studies employing these antigens showed sensitivity and specificity values above 90%, greatly impacting diagnostic accuracy. Nevertheless, despite the good results found, it is still possible to observe some bottlenecks in the development of new antigens, such as the scarcity of tests with sera from the acute phase and the variability of results in different geographic areas. In this sense, aiming to contribute to control and health programs, the continuous search for a more accurate serological diagnosis is essential, both for the acute and chronic phases of the disease.
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l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.
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Asparaginase , Phaseolus , Proteínas Recombinantes , Asparaginase/química , Asparaginase/farmacologia , Asparaginase/genética , Asparaginase/imunologia , Phaseolus/química , Humanos , Cinética , Proteínas Recombinantes/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Leucemia/tratamento farmacológico , Células K562 , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Introduction: This study in Argentina evaluated the impact of the growzen™ buddy smartphone app on adherence to recombinant human growth hormone (r-hGH) treatment. Methods: The adherence data, invitation dates with a link to the app, app activation dates, and height measurements entered were extracted from the growzen™ digital health ecosystem. Patients with 12 months of adherence data, aged ≥2 years at treatment start, and aged <19 years were selected both before and after app implementation. Mean adherence was classified as optimal (≥85%) versus suboptimal (<85%). Adherence before and after implementation and the pre-post effect on adherence were assessed. Results: Data for 830 patients were available. Prior to app implementation, the proportion of patients with optimal adherence was 68% (n = 348/515). Following the app implementation, out of 315 patients, 302 (96%) received an invitation with a link to the app, 225 (71%) activated their account, and 127 (40%) entered height data in the first year. There was a significant early increase in the proportion of patients with optimal adherence following implementation: 82% (n = 258/315), p < 0.001. After implementation, the proportion of patients with optimal adherence included 80% (n = 78/98) of those with an active account who did not enter height measurements and 89% (n = 113/127) of those who did. There was a significant and positive pre-post app effect on adherence (p < 0.01) in patients with an active account. Discussion: Our results show that using the growzen™ buddy app has a rapid and positive impact on adherence to r-hGH treatment, and patients who were more engaged with the app demonstrated better adherence.
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Hormônio do Crescimento Humano , Adesão à Medicação , Aplicativos Móveis , Proteínas Recombinantes , Smartphone , Humanos , Argentina , Masculino , Feminino , Estudos Retrospectivos , Hormônio do Crescimento Humano/uso terapêutico , Hormônio do Crescimento Humano/administração & dosagem , Adesão à Medicação/estatística & dados numéricos , Adolescente , Criança , Proteínas Recombinantes/uso terapêutico , Pré-Escolar , Adulto Jovem , AdultoRESUMO
The COVID-19 pandemic has been marked by novel viral variants, posing challenges to global public health. Recombination, a viral evolution mechanism, is implicated in SARS-CoV-2's ongoing evolution. The XBB recombinant lineage, known for evading antibody-mediated immunity, exhibits higher transmissibility without increased disease severity. We investigated the prevalence and genomic features of XBB in SARS-CoV-2-positive cases in Rio Grande do Sul (RS), Brazil. We sequenced 357 samples from epidemiological weeks (EW) 47/2022 to 17/2023, and included 389 publicly available sequences. Clinical and epidemiological data were obtained from DATASUS, e-SUS, and SIVEP GRIPE (data recording systems of the Brazilian Ministry of Health). Of these, 143 were classified as XBB and 586 were other Omicron lineages. In March 2023 (EW 10), XBB became dominant, accounting for 83.3% of cases. 97.7% of XBB-infected patients successfully recovered from the infection, with a low mortality rate (2.3%). Even after receiving three vaccine doses and having been previously infected, 59.5% of the patients experienced reinfection with XBB. However, for 54% of the individuals, the interval between their XBB infection and the last vaccine dose exceeded one year, potentially leading to a decline in antibody levels. In addition, we identified 90 mutations in RS circulating XBB, spread throughout the genome, notably in the Spike protein region associated with immune resistance. This study provides insights into the dynamics and impact of a recombinant variant becoming predominant for the first time in the state. Continued surveillance of SARS-CoV-2 genomic evolution is crucial for effective public health management.
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Vacinas contra COVID-19 , COVID-19 , Genoma Viral , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Brasil/epidemiologia , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/virologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Idoso , Filogenia , Adulto Jovem , Adolescente , Genômica/métodosRESUMO
OBJECTIVES: Exophytic Sinonasal Papilloma (ESP) is a benign tumor of the sinonasal tract. Complete surgical excision by endoscopic surgery is the treatment of choice. However, a high recurrence rate (36% at 5-year follow-up) is associated with this method, which may indicate the presence of microorganisms such as Human Papillomavirus (HPV). It is important to note that the standard treatment for ESP does not include antiviral drugs. In our study, we are testing the effectiveness of an interferon-containing drug in reducing recurrence and postoperative reactions in patients with ESP. METHODS: We included 78 patients aged 23-83 years with a confirmed diagnosis of ESP by rhinoscopy and nasal endoscopy and a positive PCR test for HPV in nasal scrapings. To compare the results, we divided the patients into main and control groups. The main group received recombinant human interferon after surgery, while the control group did not receive the drug. We performed a statistical analysis to compare the proportion of patients without reactive manifestations at different stages of the postoperative period, as well as to compare the proportion of patients with recurrent ESP at certain stages of observation. RESULTS: The introduction of recombinant human interferon accelerated the resolution of postoperative reactions and promoted the healing of the nasal mucosa after surgical removal of the ESP. We also found a statistically significant association between treatment with recombinant interferon and a reduction in the recurrence rate of ESP. CONCLUSION: According to the results of the study, it was found that in the main group of patients who received rhIFN-α2b (recombinant human Interferon alpha 2b) in the postoperative period, the frequency of relapses of ESP and the time of postoperative recovery were significantly lower than in patients in the control group who did not take the drug. LEVEL OF EVIDENCE: Cohort Study.
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Interferon alfa-2 , Interferon-alfa , Papiloma , Infecções por Papillomavirus , Humanos , Pessoa de Meia-Idade , Adulto , Idoso , Masculino , Feminino , Interferon alfa-2/uso terapêutico , Infecções por Papillomavirus/tratamento farmacológico , Idoso de 80 Anos ou mais , Adulto Jovem , Interferon-alfa/uso terapêutico , Resultado do Tratamento , Papiloma/tratamento farmacológico , Papiloma/cirurgia , Papiloma/virologia , Neoplasias Nasais/cirurgia , Neoplasias Nasais/tratamento farmacológico , Neoplasias Nasais/virologia , Proteínas Recombinantes/uso terapêutico , Recidiva Local de Neoplasia , Antivirais/uso terapêuticoRESUMO
The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85 %, 29 %, 58 %, and 31 %, respectively, and a specificity of 97.5 %, 98 %, 100 %, and 97.5 %, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95 %, 74 %, 67 %, and 52 %, respectively, and a specificity of 100 %, 99 %, 98 %, and 86 %, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.
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Anticorpos Antiprotozoários , Antígenos de Protozoários , Ensaio de Imunoadsorção Enzimática , Leishmania , Leishmaniose Cutânea , Proteínas de Protozoários , Proteínas Recombinantes , Sensibilidade e Especificidade , Humanos , Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/urina , Proteínas de Protozoários/urina , Proteínas de Protozoários/imunologia , Antígenos de Protozoários/urina , Antígenos de Protozoários/imunologia , Leishmania/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/urina , Adulto , Feminino , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/urina , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Adolescente , Idoso , Urina/química , Urina/parasitologia , Criança , Pré-Escolar , Epitopos de Linfócito B/imunologiaRESUMO
BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.
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Anticorpos Antiprotozoários , Antígenos de Protozoários , Doença de Chagas , Doenças do Cão , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Testes Sorológicos , Trypanosoma cruzi , Animais , Cães , Doença de Chagas/diagnóstico , Doença de Chagas/veterinária , Doença de Chagas/parasitologia , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/genética , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Anticorpos Antiprotozoários/sangue , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genéticaRESUMO
Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: ⢠Several T- and B-cell epitopes were identified in all the three proteins. ⢠Four different adjuvants were used in vaccine formulations. ⢠Immunization stimulated significant levels of IgG2/3 in vaccinated animals.
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Anticorpos Antibacterianos , Vacinas Bacterianas , Leptospirose , Lipoproteínas , Animais , Leptospirose/prevenção & controle , Leptospirose/imunologia , Lipoproteínas/imunologia , Lipoproteínas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/genética , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Cricetinae , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Adjuvantes Imunológicos/administração & dosagem , Imunoglobulina G/sangue , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/genética , Leptospira interrogans/imunologia , Leptospira interrogans/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vacinação , Imunidade Humoral , Leptospira/imunologia , Leptospira/genética , Imunogenicidade da VacinaRESUMO
The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.