Efficient Induction of Mouse Immune Responses to Hepatitis C Virus by Viral Core Protein-Carrying AttenuatedSalmonella typhimurium

Abstract

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for inclusion in a protective vaccine. However, this protein may attenuate the development of systemic immune responses because of its immunomodulatory properties. In this study, a eukaryotic expression plasmid, pCI-C, and an in vivo-inducible prokaryotic expression plasmid, pZW-C, for HCV core protein were constructed and transformed into attenuated Salmonella typhimurium aroA strain SL7207. The recombinant expression plasmids SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and immune responses specific to core protein were assessed. Immunization with bacteria SL7207/pCI-C led to a persistent decrease in the percentage of CD3(+)CD4(+) T cells and triggered weak anti-core IgG production. Splenocytes from SL7207/pCIC-immunized mice developed relatively weak proliferation response, low interferon-gamma secretion, and inferior cytotoxic activity compared with those from mice immunized with SL7207/pZW-C. Boost immunization with SL7207/pCI-C yielded limited improvement in the immune response, whereas boost with bacteria SL7207/pZW-C enhanced immune responses significantly. These results suggest that de novo host synthesis of native HCV core protein may cut down the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.

Plasmids Enriched with CpG Motifs Activate Human Peripheral Blood Mononuclear CellsIn Vitroand Enhance Th-1 Immune Responses to Hepatitis B Surface Antigen in Mice

“…Cell proliferation was assayed using the CellTiter 96 AQueous One Solution. The stimulation index (SI) was determined as described previously (33). PBMCs at a concentration of 2Â10 6 /mL were cultured in complete RPMI 1640 medium in the presence of various plasmids (final concentration 10 mg/mL) in a 5% CO 2 -humidified incubator at 378C.…”

“…Serial dilutions of the effector cells were cultured with 2Â10 3 SP20/S target cells in 96-well round-bottom plates and incubated for 6 h at 378C with 5% CO 2 . HBsAg-specific CTL activity was measured by lactate dehydrogenase (LDH) release using the nonradioactive cytotoxicity assay kit (Cytotox 96; Promega Corp., Madison, WI) as described previously (33). The OD 492 absorbance values of the culture supernatants were measured on an ELISA microplate reader, and cell-specific lysis was calculated according to the following formula: (experimental release -spontaneous release) / (total releasespontaneous release)Â100%.…”

The Immunogenicity in Mice of HCV Core Delivered as DNA Is Modulated by Its Capacity to Induce Oxidative Stress and Oxidative Stress Response

“…We have shown that plasmids directing higher levels of core expression were less immunogenic than directing lower levels of HCV core expression [5]. Several studies observed that transient expression of HCV core interferes with the induction of cellular immune responses against co-delivered genes [15,16]. In lines with these findings, mice transgenic for HCV core demonstrated significantly diminished T-lymphocyte responses compared to their non-transgenic littermates [17], and could not adequately respond to viral infections [18,19].…”

“…Safety of HCV core preparations in the human trials paved the way to the continuous efforts to improve its immunogenic performance. Promising results were obtained with the chimeras of HCV core protein with HBV precore regions [47], tetanus toxoid [48], HCV core carried by Lambda phage nanoparticles [49] or an attenuated Salmonella strain [15], demonstrating the possibility to overcome core-driven immune suppression.…”

Vacinação de potros com linhagem atenuada de Salmonella enterica Typhimurium carreando o gene vapA de Rhodococcus equi: avaliação clínica e imunitária

“…Assim sendo, os genes heterólogos são clonados em plasmídios asd positivos e estes, utilizados para transformar células ∆asd de Salmonella. Animais imunizados com tais linhagens recombinantes são colonizados somente por bactérias contendo o plasmídio e, portanto, expressando o antígeno heterólogo (BRANGER et al, 2009).Linhagens atenuadas de Salmonella são ótimas candidatas a vacinas orais, via pela qual apresenta muitas vantagens de aplicação em relação a outras(LIAO et al, 2007;LI et al, 2008;WANG et al, 2009), e que vem sendo constante foco de estudo(SMOLEN et al, 2010). Após administração oral, linhagens de S. enterica aderem e invadem células da mucosa intestinal, preferencialmente células M (JONES; GHORI; FALKOW, 1994), e atingem os sítios linfóides das placas de Peyer (PP) e/ouGALT (CARTER;COLLINS, 1974;HUANG;HAJISHENGALLIS;MICHALEK, 2000).…”