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1.
Front Bioeng Biotechnol ; 12: 1333548, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38449674

RESUMO

The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms.

2.
ALTEX ; 39(1): 113-122, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34798660

RESUMO

The pharmacopeia mouse neutralization assay (PMNA) is the standard method for determining the potency of phar­maceutical botulinum antitoxins. However, a PMNA requires a large number of mice, and, thus, an alternative in vitro method to replace it is needed. Herein, we developed an in vitro SiMa cell line-based neutralization assay (SBNA), compatible with a PMNA design, for therapeutic antitoxins against type E botulinum neurotoxin (BoNT/E). The SBNA measures the residual cellular activity of BoNT/E following antitoxin neutralization in the SiMa lysate using a specific quantitative sandwich ELISA for its cleaved cellular target protein SNAP-25. The potencies of different pharmaceutical antitoxin preparations were determined by applying two different quantification approaches: (1) a cutoff value, in accor­dance with the pharmacopeia concept, and (2) nonlinear regression of a standard curve generated by serial dilutions of a standard antitoxin. Both approaches achieved accurate potencies compared to the PMNA (average %RE of ~16%). Furthermore, the SBNA was able to determine in vitro, for the first time, the accurate neutralizing activity (%RE ≤ 20) of next-generation equine and rabbit therapeutic antitoxins. Collectively, a high correlation between SBNA and PMNA results was obtained for all antitoxin preparations (r = 0.99, P < 0.0001 for the standard curve approach, and r = 0.97, p < 0.0001 for the cutoff approach). In conclusion, the SBNA can potentially replace the PMNA and markedly reduce the need for laboratory animals for the approval of botulinum antitoxin preparations.


Assuntos
Antitoxinas , Toxinas Botulínicas Tipo A , Botulismo , Preparações Farmacêuticas , Alternativas aos Testes com Animais , Animais , Antitoxina Botulínica , Cavalos , Camundongos , Coelhos
3.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072087

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Descoberta de Drogas , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Animais , Antivirais/uso terapêutico , Ácido Aurintricarboxílico/farmacologia , Ácido Aurintricarboxílico/uso terapêutico , COVID-19/virologia , Chlorocebus aethiops , Ácido Elágico/farmacologia , Ácido Elágico/uso terapêutico , Heparina/farmacologia , Heparina/uso terapêutico , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos
4.
Vaccine ; 35(52): 7213-7216, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29174678

RESUMO

Botulism therapy relies on passive immunization with antitoxin. The mouse neutralization test is the only pharmacopeia assay to measure the potency of antitoxin preparations. Herein, we present an in vitro cell-based assay for the measurement of pharmaceutical type A antitoxin potency. Accuracy, reproducibility and compatibility with the mouse bioassay were demonstrated using different batches of standard antitoxin and toxin preparations. The established assay may substantially reduce the use of laboratory animals in the process of pharmaceutical antitoxin production.


Assuntos
Bioensaio/métodos , Antitoxina Botulínica/metabolismo , Técnicas In Vitro/métodos , Animais , Antitoxina Botulínica/imunologia , Toxinas Botulínicas/imunologia , Botulismo/prevenção & controle , Confiabilidade dos Dados , Camundongos , Testes de Neutralização/métodos , Reprodutibilidade dos Testes , Toxinas Biológicas
5.
Toxins (Basel) ; 8(10)2016 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-27669303

RESUMO

Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.


Assuntos
Anticorpos Neutralizantes/imunologia , Bioensaio , Antitoxina Botulínica/imunologia , Toxinas Botulínicas Tipo A/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Antitoxina Botulínica/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Catálise , Endopeptidases/metabolismo , Técnicas In Vitro , Camundongos , Peptídeos/imunologia , Peptídeos/metabolismo
6.
PLoS One ; 9(1): e87089, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24475231

RESUMO

Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50-500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Botulínicas/imunologia , Clostridium botulinum/química , Bandas Oligoclonais/imunologia , Análise de Variância , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Toxinas Botulínicas/antagonistas & inibidores , Meios de Cultura , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/metabolismo , Camundongos , Testes de Neutralização
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