RESUMO
We have previously shown that DNA vaccines expressing codon-optimized alphavirus envelope glycoprotein genes protect both mice and non-human primates from viral challenge when delivered by intramuscular electroporation (IM-EP). To determine if we could achieve equivalent immunogenicity and protective efficacy in the absence of electroporation, we co-delivered our Venezuelan equine encephalitis virus (VEEV) DNA vaccine with DNA plasmids expressing genetic adjuvants designed to augment immune responses. We tested the Th1-inducing cytokine IL-12 as well as the granulocyte growth factor GM-CSF, both of which have demonstrated significant adjuvant effect when included in clinical DNA vaccine formulations. Additionally, as multiple reports have described the necessity of IFN-αß in DNA vaccine immunogenicity, we tested vaccine plasmids encoding a potent stimulator of the IFN-αß pathway. Our data suggest that IM vaccination of mice with plasmid DNA encoding genetic adjuvants enhances VEEV vaccine immunogenicity, resulting in improved T cell responses, as well as skewing of the anti-VEEV IgG antibody isotype. Additionally, IM vaccination of VEEV DNA vaccine and IL-12 provided complete protection against aerosol VEEV challenge. Overall, our data suggest that co-delivery of genetic adjuvants with alphavirus DNA vaccines using IM delivery can influence the type of immune response obtained and provide comparable protective immunity to that achieved by IM-EP delivery of the vaccine without adjuvants.
Assuntos
Adjuvantes Imunológicos , Encefalomielite Equina Venezuelana/prevenção & controle , Imunogenicidade da Vacina , Interleucina-12/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Injeções Intramusculares , Interleucina-12/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.
Assuntos
Antivirais/metabolismo , Bunyaviridae/fisiologia , Mononegavirais/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Animais , Bunyaviridae/efeitos dos fármacos , Chlorocebus aethiops , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Mononegavirais/efeitos dos fármacos , Células VeroRESUMO
Hantaviruses cause two important human illnesses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Both syndromes are believed to be immune-mediated diseases. Monocytes/macrophages are thought to be the main target cells for hantaviruses and important sources of and targets for cytokines/chemokines secretion. THP-1 cells have been used extensively as models for primary monocytes in biocompatibility research. The aim of our study was to determine if hantaviruses induce the same immunoreactions in THP-1 cells and primary monocytes/ macrophages and might therefore be suitable for immune studies of hantaviral infections. For that purpose we compared various cytokines/chemokines and their receptors in THP-1 cell line and primary monocytes/macrophages. Infected primary monocytes/macrophages induced mostly beta-chemokines and their receptors. In contrast, THP-1 cells, expressed receptors for CXC chemokines. Surprisingly, infected macrophages underwent morphological changes toward dendritic-like cells and increased expression of co-stimulatory molecules: CD40, CD80, CD83 and CD86. Our data indicate that THP-1 cells are not ideal for in vitro research of the immunopathogenesis of hantaviruses in humans. Further, our studies revealed potential roles for cytokines/chemokines in HFRS/HPS immunopathogenesis and point to intriguing possibilities for the possible differentiation of infected macrophages to dendritic-like cells.