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1.
N Biotechnol ; 72: 11-21, 2022 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-35953030

RESUMO

Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris. The RBD was modified by adding flexible N- and C-terminal amino acid extensions that modulate protein/protein interactions and facilitate protein purification. A fed-batch methanol fermentation with a yeast extract-based culture medium in a 50 L fermenter and an immobilized metal ion affinity chromatography-based downstream purification process yielded 30-40 mg/L of RBD. Correct folding of the purified protein was demonstrated by mass spectrometry, circular dichroism, and determinations of binding affinity to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD antigen also exhibited high reactivity with sera from convalescent individuals and Pfizer-BioNTech or Sputnik V vaccinees. Immunization of mice and non-human primates with 50 µg of the recombinant RBD adjuvanted with alum induced high levels of binding antibodies as assessed by ELISA with RBD produced in HEK293T cells, and which inhibited RBD binding to ACE2 and neutralized infection of VeroE6 cells by SARS-CoV-2. Additionally, the RBD protein stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα secretion in splenocytes and lung CD3+-enriched cells of immunized mice. The data suggest that the RBD recombinant protein produced in yeast P. pastoris is suitable as a vaccine candidate against COVID-19.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Células HEK293 , Pandemias/prevenção & controle , Glicoproteína da Espícula de Coronavírus , Camundongos , Primatas
2.
Prep Biochem Biotechnol ; 47(9): 889-900, 2017 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-28816622

RESUMO

Recombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8 M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5 ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine.


Assuntos
Interleucina-15/genética , Macaca fascicularis/genética , Vacinas Sintéticas/genética , Animais , Linhagem Celular , Clonagem Molecular/métodos , Escherichia coli/genética , Humanos , Interleucina-15/imunologia , Macaca fascicularis/imunologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia
3.
Data Brief ; 4: 468-73, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26306321

RESUMO

CIGB-552 is a second generation antitumor peptide that displays potent cytotoxicity in lung and colon cancer cells. The nuclear subproteome of HT-29 colon adenocarcinoma cells treated with CIGB-552 peptide was identified and analyzed [1]. This data article provides supporting evidence for the above analysis.

4.
J Proteomics ; 126: 163-71, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26013411

RESUMO

The second generation peptide CIGB-552 has a pro-apoptotic effect on H460 non-small cell lung cancer cells and displays a potent cytotoxic effect in HT-29 colon adenocarcinoma cells though its action mechanism is ill defined. Here, we present the first proteomic study of peptide effect in HT-29 cells using subcellular fractionation, protein and peptide fractionation by DF-PAGE and LC-MS/MS peptide identification. In particular, we explored the nuclear proteome of HT-29 cells at a 5h treatment identifying a total of 68 differentially modulated proteins, 49 of which localize to the nucleus. The differentially modulated proteins were analyzed following a system biology approach. Results pointed to a modulation of apoptosis, oxidative damage removal, NF-κB activation, inflammatory signaling and of cell adhesion and motility. Further Western blot and flow-cytometry experiments confirmed both pro-apoptotic and anti-inflammatory effects of CIGB-552 peptide in HT-29 cells.


Assuntos
Adenocarcinoma , Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Neoplasias do Colo , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Proteômica , Transdução de Sinais/efeitos dos fármacos
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