Active immunization combined with cisplatin confers enhanced therapeutic protection and prevents relapses of HPV-induced tumors at different anatomical sites.

The active immunotherapy concept relies on the use of vaccines that are capable of inducing antitumor immunity, reversion of the suppressive immunological environment, and long-term memory responses. Previously, antitumor vaccines based on a recombinant plasmid (pgDE7h) or a purified protein (gDE7) led to regression of early-established human papillomavirus (HPV)-associated tumors in a preclinical model. In this work, the anticancer vaccines were combined with cisplatin to treat HPV-induced tumors at advanced growth stages. The antitumor effects were evaluated in terms of tumor regression, induction of specific CD8+ T cells, and immune modulation of the tumor microenvironment. Acute toxicity induced by the treatment was measured by weight loss and histological alterations in the liver and kidneys. Our results revealed that the combination of cisplatin with either one of the tested immunotherapies (pgDE7h or gDE7) led to complete tumor regression in mice. Also, the combined treatment resulted in synergistic effects, particularly among mice immunized with gDE7, including activation of systemic and tumor-infiltrating E7-specific CD8+ T cells, tumor infiltration of macrophages and dendritic cells, and prevention of tumor relapses at different anatomical sites. Furthermore, the protocol allowed the reduction of cisplatin dosage and its intrinsic toxic effects, without reducing antitumor outcomes. These results expand our knowledge of active immunotherapy protocols and open perspectives for alternative treatments of HPV-associated tumors.

Expressão e caracterização da glicoproteína D do HSV-1 geneticamente fusionada às oncoproteínas E6 e E7 do HPV-16 e HPV-18 (gDE7E6) em células de mamífero / Expression and characterization of the genetically fused HSV-1 glycoprotein D to E6 and E7 oncoproteins HPV-16 e HPV-18 (gDE7E6) in mammalian cells

O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais

Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes