RESUMO
New vaccine platforms are needed to address the time gap between pathogen emergence and vaccine licensure. RNA-based vaccines are an attractive candidate for this role: they are safe, are produced cell free, and can be rapidly generated in response to pathogen emergence. Two RNA vaccine platforms are available: synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA). sa-RNA is virally derived and encodes both the antigen of interest and proteins enabling RNA vaccine replication. Both platforms have been shown to induce an immune response, but it is not clear which approach is optimal. In the current studies, we compared synthetic mRNA and sa-RNA expressing influenza virus hemagglutinin. Both platforms were protective, but equivalent levels of protection were achieved using 1.25 µg sa-RNA compared to 80 µg mRNA (64-fold less material). Having determined that sa-RNA was more effective than mRNA, we tested hemagglutinin from three strains of influenza H1N1, H3N2 (X31), and B (Massachusetts) as sa-RNA vaccines, and all protected against challenge infection. When sa-RNA was combined in a trivalent formulation, it protected against sequential H1N1 and H3N2 challenges. From this we conclude that sa-RNA is a promising platform for vaccines against viral diseases.
Assuntos
Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , RNA Viral/imunologia , Animais , Modelos Animais de Doenças , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Imunização Secundária , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Viral/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
A set of 6-substituted quinolone nucleosides linked to aniline or phenol via N or O heteroatom-bridges presenting new compounds were synthesized by palladium-catalyzed Buchwald-Hartwig cross-coupling reactions. 6-Bromoquinolone nucleoside precursors, being protected by either benzoyl or TBDMS protecting groups on the ribose moiety, were subjected to different Buchwald-Hartwig conditions as the key step. Defined deprotection steps led, in good yields, to the final target compounds that carry, in position 3, either ester, acid, or amide functions. Thus, a series of novel quinolone nucleoside derivatives was obtained via a convergent synthesis route. Biological tests in human chronic myelogenous leukemia K562 cells exerted an efficient antiproliferative activity for two of them without induction of differentiation. These novel nucleosides deserve further experiments to determine their antiproliferative effects on other CML cell lines.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Nucleosídeos/farmacologia , Quinolonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Nucleosídeos/síntese química , Nucleosídeos/química , Quinolonas/síntese química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
Stille Coupling is a versatile C-C bond forming reaction with high functional group tolerance under mild conditions. Our on column synthesis concept for RNA modification is based on the incorporation of iodo substituted nucleotide precursors to RNA during automated standard solid phase synthesis via TBDMS-, TC-, and ACE- protecting group strategies. Subsequently, the RNA, still bound on solid support, is ready for orthogonal postsynthetic functionalization via Stille cross-couplings utilizing the advantages of solid phase synthesis. Several monomer test reactions were employed with 2-iodo adenosine and 5-iodo uridine and organostannanes as coupling partners under different conditions, changing the catalyst/ligand system, temperature, and reaction time as well as conventional heating and microwave irradiation. Finally, Stille cross-couplings under optimized conditions were transferred to fully protected 5-mer and 12-mer RNA oligonucleotides on-column. Deprotection and cleavage from solid support resulted in site-specifically labeled oligonucleotides. Derivatizations via Stille cross-couplings were performed initially with vinyltributylstannane as well as later with 2-furanyl-, 2-thiophene-, and benzothiophene-2-tributylstannanes yielding fluorescently functionalized RNA.
Assuntos
RNA/química , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.