An isotope dilution mass spectrometry assay to track Norovirus-like particles in vaccine process intermediates by quantifying capsid protein VP1.

The coronavirus disease (COVID-19) pandemic shows the rapid pace at which vaccine development can occur which highlights the need for more fast and efficient analytical methodologies to track and characterize candidate vaccines during manufacturing and purification processes. The candidate vaccine in this work comprises plant-derived Norovirus-like particles (NVLPs) which are structures that mimic the virus but lack any infectious genetic material. Presented here is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the quantification of viral protein VP1, the main component of the NVLPs in this study. It combines isotope dilution mass spectrometry (IDMS) with multiple reaction monitoring (MRM) to quantify targeted peptides in process intermediates. Multiple MRM transitions (precursor/product ion pairs) for VP1 peptides were tested with varying MS source conditions and collision energies. Final parameter selection for quantification includes three peptides with two MRM transitions each offering maximum detection sensitivity under optimized MS conditions. For quantification, a known concentration of the isotopically labeled version of the peptides to be quantified was added into working standard solutions to serve as an internal standard (IS); calibration curves were generated for concentration of native peptide vs. the peak area ratio of native-to-isotope labeled peptide. VP1 peptides in samples were quantified with labeled versions of the peptides added at the same level as that of the standards. Peptides were quantified with limit of detection (LOD) as low as 1.0 fmol µL-1 and limit of quantitation (LOQ) as low as 2.5 fmol µL-1. NVLP preparations spiked with known quantities of either native peptides or drug substance (DS) comprising assembled NVLPs produced recoveries indicative of minimal matrix effects. Overall, we report a fast, specific, selective, and sensitive LC-MS/MS strategy to track NVLPs through the purification steps of the DS of a Norovirus candidate vaccine. To the best of our knowledge, this is the first application of an IDMS method to track virus-like particles (VLPs) produced in plants as well as measurements performed with VP1, a Norovirus capsid protein.

Oral and nasal vaccination: current prospects, challenges, and impact of nanotechnology-based delivery systems

Abstract Currently, mucosal vaccine administration has stood out as an easier and non-invasive application method. It can also be used to induce local and systemic immune responses. In the COVID-19 pandemic context, nasal and oral vaccines have been developed based on different technological platforms. This review addressed relevant aspects of mucosal vaccine administration, with emphasis on oral and nasal vaccinations, in addition to the importance of using nanotechnology-based delivery systems to enable these strategies.

Mechanical properties and immunotherapeutic effects of dissolving microneedles with different drug loadings based on hyaluronic acid

Abstract Improving vaccine immunity and reducing antigen usage are major challenges in the clinical application of vaccines. Microneedles have been proven to be painless, minimally invasive, highly efficient, and have good patient compliance. Compared with traditional transdermal drug delivery, it can effectively deliver a large-molecular-weight drug into the skin, resulting in a corresponding immune response. However, few studies have examined the relationship between microneedle loading dose and immune effects. In this study, the hyaluronic acid (HA) conical and pyramidal dissolving microneedles were prepared by the two-step vacuum drying method, respectively. The model drug ovalbumin (OVA) was added to HA to prepare dissolving microneedles with different loading amounts. The mass ratios of HA to OVA were 5:1, 5:3, and 5:5. The mechanical properties of the dissolving microneedles were characterized using nanoindentation and in vitro puncture studies. The immune effects of the matrix and drug content were studied in Sprague-Dawley (SD) rats. Finally, the diffusion behavior of OVA and the binding mode of HA and OVA in the microneedles were simulated using Materials Studio and Autodocking software. The experimental results showed that the conical microneedles exhibited better mechanical properties. When the mass ratio of HA to OVA was 5:3, the immune effect can be improved by 37.01% compared to subcutaneous injection, and achieved a better immune effect with relatively fewer drugs. This conclusion is consistent with molecular simulations. This study provides theoretical and experimental support for the drug loading and efficacy of microneedles with different drug loadings

Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology / Avaliação de três vacinas de Aeromonas hydrophila precipitadas com alúmen administradas a Labeo rohita, Cirrhinus mrigala e Ctenopharyngodon idella: imunocinética, desafio de imersão e histopatologia

Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was [...].

Aeromonas hydrophila é uma causa de surtos de doenças infecciosas em espécies de carpas cultivadas em países do sul da Ásia, incluindo o Paquistão. Essa bactéria ganhou resistência a uma ampla gama de antibióticos, e medidas preventivas robustas são necessárias para controlar sua disseminação. Nenhum uso anterior de vacinas para peixes foi relatado no Paquistão. O presente estudo tem como objetivo desenvolver e avaliar vacinas inativadas contra cepa local de A. hydrophila no Paquistão com precipitado de alúmen como adjuvante. O potencial imunogênico da vacina foi avaliado em duas carpas principais indianas (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) e uma carpa chinesa (Grass Carp: Ctenopharyngodon idella). Os peixes foram vacinados por via intraperitoneal, seguido de um desafio por imersão. Peixes com idade média de 4-5 meses foram distribuídos aleatoriamente em três grupos vacinados com três concentrações de vacina de 108, 109 e 1010 unidades formadoras de colônias (UFC) / ml e um grupo de controle. Foi aplicada dose fixa de 0,1ml em cada peixe no 1º dia e dose de reforço 15 dias pós-vacinação (DPV). Amostras de sangue foram coletadas em 14, 28, 35, 48 e 60 DPV para determinar os títulos de anticorpos no soro sanguíneo usando o teste de fixação de elogio (CFT). Os peixes foram desafiados a 60 DPV com infecciosa A. hydrophila com 108 CFU / ml por imersão. Níveis significativamente mais elevados de títulos de anticorpos foram observados em 28 DPV em todos os grupos vacinados, em comparação com aqueles no grupo de controle. Na experiência de desafio, o RPS médio (sobrevivência percentual relativa) foi de 71% para os grupos vacinados com 109 e 1010 CFU / ml e 86% para 108 CFU / ml. A vacina com 108 UFC / ml induziu a maior resposta imune seguida por 109 e 1010 UFC / ml. A resposta imune de L. rohita e C. idella foi melhor do que a de C. mrigala. Em geral, histopatologia normal foi observada em diferentes [...].

Vacinas / Vaccines

Ao longo da história, as vacinas evitaram inúmeros casos de doenças, incapacidades e salvaram milhões de vidas (FDA, 2020). Atualmente, o avanço da vacinação contra a COVID19 no Brasil permitiu alcançar notáveis ganhos em saúde pública, reduzindo de maneira significativa a ocorrência de casos graves e óbitos pela doença (BRASIL, 2021). As vacinas são consideradas a invenção médica que mais salvou vidas na história da humanidade, uma das conquistas mais significativas da ciência e da saúde pública, um dos maiores triunfos na história da medicina. Como forma de prevenção contra diferentes doenças infecciosas, as vacinas são dadas a milhões de bebês, crianças, adolescentes e adultos; para que isso possa acontecer é fundamental que sejam comprovadamente seguras e eficazes (HAN, 2015; GRUBER et al., 2018; FDA, 2020; PINHEIRO, 2021).

Throughout history, vaccines have prevented countless cases of disease, disability and saved millions of lives (FDA, 2020). Currently, the advance of vaccination against COVID19 in Brazil has made it possible to achieve notable gains in public health, significantly reducing significant occurrence of severe cases and deaths from the disease (BRASIL, 2021). Vaccines are considered the most life-saving medical invention in human history, one of the most significant achievements of science and public health, one of the greatest triumphs in the history of medicine. As a form of prevention against different infectious diseases, vaccines are given to millions of babies, children, teenagers and adults; for this to happen, it is essential that they are proven to be safe and effective (HAN, 2015; GRUBER et al., 2018; FDA, 2020; PINHEIRO, 2021).

Caracterização funcional de proteínas hipotéticas de Leptospira interrogans como adesinas e potenciais antígenos vacinais e para diagnóstico / Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.

Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

Multi-residue method for the detection of 40 ß-lactam-antibiotics in vaccines by LC-MS/MS.

Antibiotics are widely used to treat bacterial infections during the process of vaccine production and storage resulting in antibiotic residues that can cause serious harm. A simple and sensitive method for residue analysis of 40 ß-lactam antibiotics was developed and validated for vaccines including inactivated enterovirus 71 vaccine (Vero cells), recombinant hepatitis B vaccine (Saccharomyces cerevisiae), and live attenuated varicella vaccine using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI- MS/MS). Samples were prepared with acetonitrile as the protein precipitant. LC separation was performed on a C18 column. These analytes were determined by LC-MS/MS operating multiple-reaction monitoring (MRM) scans in positive mode. The ranges for limits of detection (LOD) and quantification (LOQ) were as follows: 0.02-4 ng/dose (S/N ≥ 3) and 0.04-10 ng/dose in inactivated enterovirus 71 vaccine (Vero cells) and recombinant hepatitis B vaccine (Saccharomyces cerevisiae), 0.04-16 ng/dose and 0.2-20 ng/dose in live attenuated varicella vaccine. The ranges of recoveries of all antibiotics were 84.5%-108.2% in inactivated enterovirus 71 vaccine (Vero cells), 73%-108% in recombinant hepatitis B vaccine (Saccharomyces cerevisiae), and mostly 68.2%-107.8% in live attenuated varicella vaccine. This method simultaneously offers qualitative and quantitative analysis of multi-antibiotics in vaccines, which improves vaccine safety.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Vacinas Covid-19: panorama / Vaccines Covid-19: overview

Diversos países já aprovaram, em caráter emergencial ou definitivo, o uso de imunizantes para vacinação da população contra o novo coronavirus. Até o dia 05 de janeiro de 2021, ao menos 48 países já começaram a imunizar suas populações. Os últimos a entrarem na lista foram: República de Palau, Islândia, Singapura, Irlanda, Belarus e a Argentina (CNNBRASIL, 2021a). Com a vacinação da população já iniciada em diferentes países, também é relevante o acompanhamento das doses já administradas em razão da população do país e em números absolutos.

Several countries have already approved, on an emergency or definitive basis, the use of immunizations to vaccination of the population against the new coronavirus. As of January 5, 2021, at least 48 countries have begun immunizing their populations. The last to enter the list were: Republic of Palau, Iceland, Singapore, Ireland, Belarus and Argentina (CNNBRASIL, 2021a). With the vaccination of the population already initiated in different countries, it is also relevant the monitoring of doses already administered due to the population of the country and in absolute numbers.

Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze.

Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.

Covid-19: Anvisa e Fiocruz discutem registro de vacina

A Anvisa e a Fiocruz realizaram a primeira reunião para tratar do registro da vacina contra Covid-19 desenvolvida pela Universidade de Oxford em parceria com a empresa Astrazeneca do Brasil. A vacina é a ChAdOx1 nCoV-19, também conhecida como AZD1222, que obteve, em junho, a aprovação da Agência para a realização de estudos clínicos de fase III no país

Coronavírus: São Paulo procura parcerias para receber ou desenvolver vacinas

Saiba mais em www.saopaulo.sp.gov.br/coronavirus

Estratégia usada em vacina contra o ebola pode ser aplicada para o coronavírus

Em entrevista à Agência FAPESP, Edécio Cunha Neto, professor do Instituto do Coração (Incor) da Faculdade de Medicina da Universidade de São Paulo (USP), explica como a estratégia usada para desenvolver uma candidata à vacina contra o ebola pode orientar na criação de um imunizante contra o novo coronavírus SARS-CoV-2, causador da COVID-19. O artigo An effective CTL peptide vaccine for ebola Zaire based on survivors’ CD8+ targeting of a particular nucleocapsid protein epitope with potential implications for Covid-19 vaccine design, (doi.org/10.1101/2020.02.25.963546), de CV Herst, S Burkholz, J Sidney, A Sette, PE Harris, S Massey, T Brasel, E Cunha Neto, DS Rosa, WCH Chao, R Carback, T Hodge, L Wang, S Ciotlos, P Lloyd e R Rubsamen, pode ser lido no bioRxiv em www.biorxiv.org/content/10.1101/2020.02.25.963546v2.abstract. E o artigo Coronavirus infections – more than just the common cold (10.1001/jama.2020.0757), de Catharine I. Paules, Hilary D. Marston e Anthony S. Fauci, pode ser lido no Journal of the American Medical Association (JAMA) em jamanetwork.com/journals/jama/fullarticle/2759815.

Determination of glycosylation degree for glycoconjugate vaccines using a solid-phase extraction combined with liquid chromatography and tandem mass spectrometry method.

In this study, a solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed to determine the degree of glycosylation of glycosylation sites and the ratio of free carrier protein to total carrier protein for glycoconjugate vaccines. To remove and enrich the glycosylated peptides, a solid-phase extraction method was developed, optimized, and hyphenated to liquid chromatography-tandem mass spectrometry. The developed solid-phase extraction with liquid chromatography-tandem mass spectrometry method was shown to possess a wide linear dynamic range (0.03-100 µg/mL), a high sensitivity (0.03 µg/mL for CRM197), good interday and intra-day precision (relative standard deviation of peak area < 3.3%), and good recoveries from vaccine matrix (90-105%). Finally, the method was utilized to determine the degree of glycosylation and free carrier protein to total carrier protein ratio for pneumococcal conjugate vaccines and meningococcal vaccines. For quality evaluation of glycoconjugate vaccines, the method could provide more information than the traditional size exclusion chromatography method. Fourteen and twelve reported glycosylation sites for CRM197- and tetanus toxin-based vaccines can be detected, respectively.

Live from WHO Headquarters: COVID-19 daily press briefing 27 April 2020

Originally broadcast live on 27 April 2020, the daily press briefing on coronavirus COVID-19, direct from WHO Headquarters, Geneva Switzerland with Dr Tedros WHO Director-General, Dr Micheal Ryan, Executive Director of the Health Emergencies Programme, and Dr Maria Van Kerkhove, Technical lead, Health Emergencies Programme. PLEASE NOTE. Due to network problem, the livestream was cut at 55mins.

Fiocruz Minas participa de estudo para vacina contra Covid-19

Pesquisadores da Fiocruz Minas estão integrando uma rede do Instituto Nacional de Ciência e Tecnologia em Vacinas (INCTV) para o desenvolvimento de uma vacina contra o novo coronavírus (Sars-CoV-2). O estudo terá como base uma técnica elaborada pelo Grupo de Imunologia de Doenças Virais da Fiocruz Minas, que utiliza o vírus da influenza para gerar resposta imunológica contra o novo coronavírus.

Comparison of Limulus Amoebocyte Lysate and Recombinant Factor C Assays for Endotoxin Detection in Four Human Vaccines with Complex Matrices.

Endotoxins, heat-stable lipopolysaccharides from Gram-negative bacteria, are potential contaminants that can be introduced during manufacturing of pharmaceutical products, including vaccines. Parental pharmaceutical products undergo endotoxin testing because endotoxins are pyrogenic in humans and can induce severe physiological reactions. Currently, animal-derived Limulus amoebocyte lysate (LAL) assays are widely used. Assays using recombinant factor C (rFC), a nonanimal-derived reagent, have been proposed as alternatives. Some components in the matrices of pharmaceutical products can interfere with these assays. We compared two LAL- and two rFC-based assays for endotoxin detection in four complex human vaccine matrices. We showed that the results for the rFC-based assays were at least equivalent to those for the LAL-based assays, although the rFC-based assays were found to be adequate but slightly less suitable for one of the products that contained proteases as the methods used to inactivate the proteases reduced the assay performance. Likewise, LAL was adequate but less suitable for another product that contained glucans. The rFC assays offer a number of benefits, including compliance with the principles of the 3Rs, i.e., replacement, reduction, and refinement of animal testing by safeguarding animal welfare and promoting more ethical and sustainable use of animals for testing. After they are fully validated, as per the compendial requirements, they could be considered as suitable replacement assays for the detection of endotoxin in the manufacturing processes of pharmaceutical products. In summary, we demonstrated that both LAL and rFC assays are adequate for testing and releasing four vaccine products.

[Aluminium adjuvant exposure through vaccines in France in 2018]. / Exposition à l'aluminium vaccinal en France en 2018.

OBJECTIVES: Aluminum-containing vaccine adjuvants stimulate an adequate immune response to vaccination. The safety and rapid elimination of these molecules, a guarantee of their safe use for several decades, have been challenged by a growing number of studies over the last 20 years. Evaluation of exposure to aluminum adjuvants of an individual is thus essential. The current review answers the following questions: what is the exposure of aluminum adjuvants of an individual vaccinated in France? What are the factors of variation? METHODS: To evaluate the immunization exposure to aluminum for a vaccinee in France, we used the 2018 vaccination schedule and the Social Security database for vaccines reimbursed that year. French mandatory and recommended vaccines for an individual who does not travel abroad and has no particular professional obligations have been taken into account. RESULTS: Our results show that an individual following the vaccination requirements and recommendations of 2018 receives between 2545 and 7735µg of Al3+ during his lifetime, and at least 50% before the age of 1year. Exposure varies with age, weight, sex, and choice of administered vaccines. CONCLUSION: Vaccines with higher doses of aluminum are mainly injected at the beginning of life. Women receive a proportionately larger dose than men. The most reimbursed vaccines are often those with the highest amount of aluminum salts.

Schistosomiasis and hookworm infection in humans: Disease burden, pathobiology and anthelmintic vaccines.

Helminth diseases are the ancient scourges of humans and their damages are 'silent and insidious'. Of the helminth infections, schistosomiasis and hookworm infection have a great impact. This review covers information regarding vaccine candidates against schistosomiasis and hookworms that reached at least up to the phase-1 trial and literatures regarding other vaccine candidates have been excluded. For clinical manifestations, all available literatures were included, and for epidemiology and global burden of the diseases (GBD), literatures only within 2000-2019 were included. Literatures were searched surfing various databases including PubMED, Google Scholar, and Science Direct and overall over 150 literatures were identified. Globally ~250 million people are suffering from schistosomiasis, resulting 1430 thousand DALY (disability adjusted life year) per year. On the other hand, about 1.3 billion people are infected with hookworm (HW), and according to WHO, ~878 million school-age children (SAC) are at risk. HW is estimated to cause 65,000 deaths annually, accounts for 845 thousand DALYs as well as to cause 6-35.3% loss in productivity. Despite tremendous efforts, very few anthelmintic vaccine candidates such as Na-GST-1, Na-APR-1 and Na-ASP-2 against HW, and Sm28GST/Sh28GST, Sm-p80, Sm14 and Sm-TSP-1/SmTSP-2 against schistosomiasis reached up to the clinical trials. More efforts are needed to achieve the WHO targets taken against the maladies.

Characterization of a chymotrypsin-like enzyme from Trichinella spiralis and its facilitation of larva penetration into the host's enteral epithelial cells.

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis.