RESUMO
We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.
Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas/química , Animais , Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Células Sanguíneas/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Cobaias , Haplorrinos , Humanos , Membranas Artificiais , CoelhosRESUMO
Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA(63)). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus alpha-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (approximately 1400 g/mol). The results suggest that the limiting diameter of the PA(63) pore is <2 nm, which is consistent with an all-atom model of the PA(63) channel and previous experiments using large ions.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/ultraestrutura , Bacillus anthracis/química , Toxinas Bacterianas/química , Modelos Químicos , Modelos Moleculares , Polietilenoglicóis/química , Simulação por Computador , Eletrólitos/química , Porosidade , Conformação ProteicaRESUMO
Filoviruses are causative agents of hemorrhagic fever, and to date no effective vaccine or therapeutic has been approved to combat infection. Filovirus glycoprotein (GP) is the critical immunogenic component of filovirus vaccines, eliciting high levels of antibody after successful vaccination. Previous work has shown that protection against both Ebola virus (EBOV) and Marburg virus (MARV) can be achieved by vaccinating with a mixture of virus-like particles (VLPs) expressing either EBOV GP or MARV GP. In this study, the potential for eliciting effective immune responses against EBOV, Sudan virus, and MARV with a single GP construct was tested. Trimeric hybrid GPs were produced that expressed the sequence of Marburg GP2 in conjunction with a hybrid GP1 composed EBOV and Sudan virus GP sequences. VLPs expressing these constructs, along with EBOV VP40, provided comparable protection against MARV challenge, resulting in 75 or 100% protection. Protection from EBOV challenge differed depending upon the hybrid used, however, with one conferring 75% protection and one conferring no protection. By comparing the overall antibody titers and the neutralizing antibody titers specific for each virus, it is shown that higher antibody responses were elicited by the C terminal region of GP1 than by the N terminal region, and this correlated with protection. These data collectively suggest that GP2 and the C terminal region of GP1 are highly immunogenic, and they advance progress toward the development of a pan-filovirus vaccine.
Assuntos
Proteção Cruzada , Ebolavirus/imunologia , Marburgvirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Ebolavirus/genética , Feminino , Cobaias , Doença pelo Vírus Ebola/prevenção & controle , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sobrevida , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Virossomos/genética , Virossomos/imunologiaRESUMO
BACKGROUND: Virus-like particle (VLP)-based vaccines have the advantage of being morphologically and antigenically similar to the live virus from which they are derived. Expression of the glycoprotein and VP40 matrix protein from Lake Victoria marburgvirus (MARV) results in spontaneous production of VLPs in mammalian cells. Guinea pigs vaccinated with Marburg virus VLPs (mVLPs) or inactivated MARV (iMARV) develop homologous humoral and T-cell responses and are completely protected from a lethal homologous MARV challenge. AIMS & METHODS: To determine whether mVLPs based on the Musoke (aka Lake Victoria) isolate of MARV could broadly protect against diverse isolates of MARV, guinea pigs were vaccinated with mVLPs or iMARV-Musoke and challenged with MARV-Musoke, -Ravn or -Ci67. RESULTS: Prior to challenge, the mVLP- and iMARV-vaccinated guinea pigs had high levels of homologous MARV-Musoke and heterologous MARV-Ravn and -Ci67 antibodies. The Musoke-based mVLPs and iMARV vaccines provided complete protection in guinea pigs against viremia, viral replication and pathological changes in tissues, and lethal disease following challenge with MARV-Musoke, -Ravn or -Ci67. Guinea pigs vaccinated with RIBI adjuvant alone and infected with guinea pig-adapted MARV-Musoke, -Ravn or -Ci67 had histopathologic findings similar to those seen in the nonhuman primate model for MARV infection. Based on the strong protection observed in guinea pigs, we next vaccinated cynomolgus macaques with Musoke-based mVLPs and showed the VLP-vaccinated monkeys were broadly protected against three isolates of MARV (Musoke, Ravn and Ci67). CONCLUSION: Musoke mVLPs are effective at inducing broad heterologous immunity and protection against multiple MARV isolates.