Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like particles.

Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.

Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles.

Filamentous phages are now the most widely used vehicles for phage display and provide efficient means for epitope identification. However, the peptides they display are not very immunogenic because they normally fail to present foreign epitopes at the very high densities required for efficient B-cell activation. Meanwhile, systems based on virus-like particles (VLPs) permit the engineered high-density display of specific epitopes but are incapable of peptide library display and affinity selection. We developed a new peptide display platform based on VLPs of the RNA bacteriophage MS2. It combines the high immunogenicity of MS2 VLPs with the affinity selection capabilities of other phage display systems. Here, we describe plasmid vectors that facilitate the construction of high-complexity random sequence peptide libraries on MS2 VLPs and that allow control of the stringency of affinity selection through the manipulation of display valency. We used the system to identify epitopes for several previously characterized monoclonal antibody targets and showed that the VLPs thus obtained elicit antibodies in mice whose activities mimic those of the selecting antibodies.

Immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus L2 epitope, on virus-like particles of the RNA bacteriophage PP7.

The immunogenicity of an antigen can be dramatically increased by displaying it in a dense, multivalent context, such as on the surface of a virus or virus-like particle (VLP). Here we describe a highly versatile VLP platform for peptide display based on VLPs of the RNA bacteriophage PP7. We show that this platform can be used for the engineered display of specific peptide sequences as well as for the construction of random peptide libraries. Peptides representing the FLAG epitope, the V3 loop of HIV gp120, and a broadly cross-type neutralizing epitope from L2, the minor capsid protein of Human Papillomavirus type 16 (HPV16), were inserted into an exposed surface loop of a form of PP7 coat protein in which the two identical polypeptides of coat were fused together to form a single-chain dimer. The recombinant proteins assembled into VLPs, displayed these peptides on their surfaces, and induced high-titer antibody responses. The single-chain dimer was also highly tolerant of random 6-, 8-, and 10-amino acid insertions. PP7 VLPs displaying the HPV16 L2 epitope generated robust anti-HPV16 L2 serum antibodies after intramuscular injection that protected mice from genital infection with HPV16 pseudovirus as well as a heterologous HPV pseudovirus type, HPV45. Thus, PP7 VLPs are well-suited for the display of a wide diversity of peptides in a highly immunogenic format.

Consequências do estresse na atividade do eixo hipotálamo-hipófise-adrenal e no envelhecimento / Consequences of stress in hypothalamic-pituitary-adrenal axis activity and agind

o estresse não é um fenômeno da vida contemporânea, mas inato ao organismo, uma vez que o primeiro ser já deveria sofrer dificuldades para manter seu equilíbrio. O assunto é muito discutido atualmente em decorrência das consequências negativas do estresse na vida diária das pessoas, tais como cansaço, irritabilidade, labilidade emocional, fragilidade imunológica, gastrite, asma. Contudo, há também os efeitos benéficos, já que o estresse é essencial à vida. O preocesso de envelhecimento sempre foi motivo de preocupação para a medicina, entretanto, das centenas de trabalhos publicados sobre as mudanças na idade avançada, não se sabe quais os mecanismos exatos que levam à senescência. todavia, nos últimos trinta anos houve progressos notáveis, sobretudo devido a trabalhos experimentais que demonstram os papéis significativos de agentes externos e internos no processo de envelhecimento. Este trabalho teve por objetivo discutir como o organismo reage aos estímulos estressores, enfatizando o eixo hipotálamo-hipófise-adrenal, as mudanças fisiológicas e bioqu´´imicas que ocorrem tanto no jovem como no idoso, o momento em que estas mudanças passam a ser prejudiciais e a sua associação com as doenças do envelhecimento. É também destacada a participação do estresse no processo normal de snescência