RESUMO
Hepatitis B core antigens (HBcAg) and hepatitis B surface antigens (HBsAg) are the main structural antigens of hepatitis B virus (HBV). Both antigens are potent immunogens for experimental animals as well as in acutely infected patients. A novel formulation based on the combination of HBsAg and HBcAg has been developed as a therapeutic vaccine candidate, aimed at inducing an immune response capable of controlling the infection. An immunization schedule was conducted to evaluate the immunogenicity of this formulation after simultaneous immunization by the intranasal and parenteral routes using different schedules and doses. Humoral and cellular immune responses generated in blood and spleen were evaluated by engyme-linked immunosorbent assay (ELISA) and enzyme-liked immunospot (ELISPOT) assays respectively. A first experiment evaluated two groups of mice simultaneously immunized by intranasal (IN) and subcutaneous (SC) routes, one including alum by SC route and, in the other, the formulation was injected without adjuvant. As a result, alum adjuvant did not increase the immunogenicity under the studied conditions. In fact, the group without alum induced the most potent immune response. The immune response was enhanced by combining IN and SC immunization compared to the SC route alone. In a second experiment, mice were immunized by different mucosal routes at the same time, and compared to the simultaneously (IN/SC) immunized groups. It was demonstrated that there is no improvement on the resulting immune response by using multiple routes of immunizations simultaneously; however, the increase of the antigen dose induced a superior immune response. Interestingly, the increase of antigen dose only by SC route did not favor the resulting immunogenicity. In conclusion, the use of HBsAg transgenic mice has proven useful to optimize the formulation, avoiding the unnecessary use of alum as adjuvant as well as provided information of the role of different mucosal immunization routes and antigen dose on the resulting immune response. How to cite this article: Trujillo H, Blanco A, García D, Freyre F, Aguiar J, Lobaina Y, Aguilar JC. Optimization of a Therapeutic Vaccine Candidate by Studying Routes, Immunization Schedules and Antigen Doses in HBsAg-positive Transgenic Mice. Euroasian J Hepato-Gastroenterol 2014;4(2):70-78.
RESUMO
From genetic material of hybridoma cells, we have generated a recombinant single-chain antibody fragment (scFv antibody) specific to carcinoembryonic antigen (CEA), which can substitute an intact murine monoclonal immunoglobulin G1 (IgG1) antibody, also developed by our group, and used in clinical practice for many years. In this paper, we examine a novel one-step method for direct 99mTc labelling of a recombinant anti-CEA scFv fragment through a C-terminal peptide tag containing a six-histidine sequence. This C-terminal peptide tag does not affect antigen binding, and was employed as a strategy for the one-step method of direct 99mTc labelling of a recombinant antibody fragment, based on the criteria of Zamora and Rhodes (Zamora PO, Rhodes BA. Imidazoles as well as thiolates in proteins bind technetium-99m. Bioconj Chem 1992; 3: 493-498). This is a novel technique for the rapid labelling of molecules, suitable for in vivo trials. The method yields >95% labelling efficiency without major effects on biological or in vitro stability.
Assuntos
Histidina , Região Variável de Imunoglobulina/química , Fragmentos de Peptídeos/química , Compostos Radiofarmacêuticos/química , Tecnécio/química , Animais , Anticorpos Monoclonais , Antígeno Carcinoembrionário/imunologia , Cromatografia em Gel , Cromatografia em Camada Fina , Cisteína , Histidina/análogos & derivados , Histidina/farmacocinética , Marcação por Isótopo , Masculino , Camundongos , Camundongos Endogâmicos , Fragmentos de Peptídeos/farmacocinética , Controle de Qualidade , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes , Tecnécio/farmacocinética , Distribuição TecidualRESUMO
In this paper we report the development of a recombinant strain of the yeast Pichia pastoris, which secretes an anti-carcinoembryonic antigen single chain Fv (scFv) antibody fragment to the culture supernatant as a biologically active protein, at levels of 1.2 g l(-1). The yeast scFv was purified by IMAC, with a final yield of approximately 0.440 g of 93% pure scFv per liter of culture supernatant. The specific activity in ELISA of the yeast scFv was almost three times higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level production of scFv antibody fragments with potential in vivo diagnostic and therapeutic applications.
Assuntos
Adenosina Trifosfatases , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Antígeno Carcinoembrionário/isolamento & purificação , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Fermentação , Regulação Fúngica da Expressão Gênica , Fragmentos de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Camundongos , Proteína MutS de Ligação de DNA com Erro de Pareamento , Pichia/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Transformação GenéticaRESUMO
A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Humanos , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/isolamento & purificação , Microscopia Eletrônica , Dados de Sequência Molecular , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The expression of chimeric genes in the mammary gland of transgenic farm animals has become an alternative for the large-scale production of recombinant proteins and for the modification of milk composition. In this paper, we show that a mouse/human chimeric antibody against the human CD6 leukocyte antigen can be assembled and correctly folded by the mammary gland, and secreted to milk, where it maintains its specificity. The base sequences encoding for the heavy and light chain variable regions of the anti-CD6 mouse monoclonal antibody IOR-T1 were cloned by the polymerase chain reaction from hybridoma cDNA, coupled to human heavy and light chain constant region genes, and inserted in a vector containing the 5' regulatory region of the rabbit whey acidic protein gene. Transgenic mice were produced by conventional pronuclei microinjection techniques. Integration and transgene copy number were determined by Southern blot. Assembled human immunoglobulin was detected in milk using a sandwich ELISA. Expression levels of chimeric antibodies in milk were determined to be around 400 micrograms/ml by Western blot, using CHO-derived chimeric IOR-T1 antibodies as reference. The chimeric antibodies produced in milk recognized human peripheral blood T lymphocytes by indirect immunofluorescence, with the classical patch-like pattern of IOR-T1.