[Study on combined immunization of rAd/MDC-VP1 and pcDNA3/MDC-VP1 against Coxsackievirus B3 challenge in mice].

AIM: To immunize the mice using the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost strategy and observe its immunological effect against Coxsackievirus B3(CVB3). METHODS: BALB/c mice were randomly divided into four groups: PBS group, rAd/MDC-VP1 group, pcDNA3/MDC-VP1 group and rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost group. Mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assays respectively. The Lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with lethal dose of CVB3, and the serum virus titer was assayed and the protection efficacy against Coxsackievirus infection was observed. RESULTS: It was observed that the titers of CVB3 VP1 specific IgG and neutralizing antibody, non-specific lymphocytic proliferation activity and specific lymphocytic CTL cytotoxic activity of the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost group were much higher than those of the rest groups(P<0.05), what's more, after CVB3 challenged, the serum virus titer of this group was lower and the protection rate(41.67%) was higher (P<0.05). CONCLUSION: Both the cellular and humoral immune responses in mice could be significantly enhanced by the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost strategy and the protection rate after challenged by lethal dose of virus could be increased.

Immunogenicity of a lentiviral-based DNA vaccine driven by the 5'LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques.

Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Δ4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Δ4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Δ4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Δ4-SHIVKU2 and CAL-Δ4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-γ ELISPOT responses in Δ4-SHIVKU2- and CAL-Δ4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

Generation of protective immune responses against coxsackievirus B3 challenge by DNA prime-protein boost vaccination.

Coxsackievirus B3 (CVB3) causes viral myocarditis and can ultimately result in dilated cardiomyopathy. However, there is no vaccine available for clinical use. In this study, we assessed the protection provided by three immunization strategies against CVB3 infection. Vaccination was performed with a DNA vaccine expressing the cloned capsid gene VP1 or a vaccine developed from purified VP1 protein. Third, a strategy of vaccination was attempted with the DNA vaccine followed by two boosts with the recombinant protein vaccine (DNA prime-protein boost vaccine). Followed immunization, mice were challenged with CVB3 infection. Improved induction of CVB3-specific antibodies and neutralizing antibodies were found in mice immunized by the DNA prime-protein boost regimen. Furthermore, virus-specific cytotoxic activity of spleen cells derived from DNA prime-protein boost vaccinated mice was elicited. In addition, the DNA prime-protein boost vaccine resulted in protection of 75% of mice from lethal CVB3 challenge and a significant reduction of viral load in sera of immunized mice after acute CVB3 infection. There was a significant reduction in myonecrosis and infiltrating myocardial immune cells indicating reduced severity of myocarditis in surviving mice. These findings demonstrated that a DNA prime-protein boost immunization strategy, but not a DNA vaccine or protein vaccine alone, was effective in eliciting both humoral and cell-mediated immune responses against CVB3 infection in mice and might be a promising vaccine candidate.

[Construction of the recombinant adenovirus expressing the CVB3 sVP1-C3d3 protein and study on its immunological effects in mice].

AIM: To construct recombinant adenovirus Ad/C3d3-sVP1 and investigate the immune effects against coxsackievirus infection in mouse. METHODS: The recombinant adenovirus Ad/sVP1-C3d3 was constructed and packaged. BALB/c mouse were divided into four groups: Ad/sVP1-C3d3 group, Ad/VP1 group, Ad group and PBS group. The mice in each group were immunized by intramuscular injection. The titers of sera IgG and neutralizing antibody were detected by ELISA method and trace neutralization assay, respectively.The specific CTL cytotoxic activity was detected by CCK-8 assay. The mice in each group were challenged with lethal dose of coxsackievirus, the titers of the sera virus were titrated. RESULTS: The recombinant adenovirus Ad/sVP1-C3d3 was successfully constructed. It's observed that the titers of CVB3 VP1 specific antibody and neutralizing antibody were much higher than those of the other three groups(P<0.01). CTL cytotoxicity activities was much higher than PBS and Ad group(P<0.01), but little higher than Ad/VP1 group(P<0.05).The titer of sera virus was lower than Ad and PBS groups after CVB3 challenged(P<0.05). CONCLUSION: Both the celluar and humoral immune responses in mice could been significantly enhanced by Ad/sVP1-C3d3.

[Immune effects of four coxsackievirus B3 VP1 DNA fusion vaccines in mice].

AIM: To compare the immunogenicity and protective effects on CVB3 infected mice of four DNA fusion vaccines coupling coxsackievirus B3 (CVB3) VP1 with macrophage-derived chemokine (MDC), C3d3, shiga toxin B subuit (STxB) and mouse beta-defensin-2 (mBD2), respectively. METHODS: BALB/c mice were divided into 6 groups randomly and inoculated in quadriceps at 3-week interval for 3 times with pcDNA3, pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1, respectively. Fourteen days after every inoculation, serum samples were collected and CVB3 specific neutralizing antibodies were determined. Three weeks after the last immunization, the mice were treated in three ways. First, the spleen cells were isolated from 3 mice of each group and specific CTL activities were tested. Second, 3 mice of each group were further challenged with 3LDLD(50); CVB3 and sacrificed 7 days later, and their blood viral titers were evaluated. Third, the rest mice of each group were subjected to intraperitoneal (i.p.) challenge with 5LDLD(50); CVB3 and their survival was observed. RESULTS: The neutralizing antibodies against CVB3 were induced in pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1 groups, and antibody titers correlated with the number of injections (P<0.01). After three immunizations, the antibody titers in pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2 -VP1 groups were higher than the ones in pcDNA3/VP1and pcDNA3/STxB-VP1 groups (P<0.01). The specific CTL activities in both pcDNA3/STxB-VP1 and pcDNA3/mBD2-VP1 groups were significantly stronger than those in the other groups (P<0.01). After CVB3 challenge, the blood viral titers in the pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2-VP1 groups were lower than those in the other groups (P<0.01), and the pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 mice survived longer than the others (P<0.05). CONCLUSION: Both pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 vaccines could induce stronger immune responses, resulting in higher survival rates and better protective effects on CVB3 infection than pcDNA3/STxB-VP1, pcDNA3/mBD2-VP1 and pcDNA3/VP1 vaccines.

Nef modulates the immunogenicity of Gag encoded in a non-infectious HIV DNA vaccine.

Gag-CD8+ T cell responses are associated with immune control of HIV infection. Since during HIV infection Nef impairs T cell responses, we evaluated whether deletion of nef from a non-infectious HIV DNA vaccine (Delta4 Nef+), creating Delta5 Nef(-), would affect its immunogenicity. When compared with Delta4, mice injected with Delta5 developed significantly lower CD8+ T cell responses to Gag, but no significant change in the responses to Env was observed. In vitro, deletion of Nef abrogated the induced cell death, production of virus-like particles and release of Gag from transfected cells. Thus, the effect of Nef in causing extrusion of Gag might adjuvant the CD8+ T cell responses to Gag in DNA vaccine.

Complete genomes of three subtype 6t isolates and analysis of many novel hepatitis C virus variants within genotype 6.

In this study, the complete genomic sequence was determined for three hepatitis C virus variants (VT21, TV241 and TV249) of genotype 6 that do not classify within the established subtypes. All three genomes were isolated from patients in Vietnam and sequenced using 100 microl of serum. They showed 91.4-93.6% nucleotide similarities to each other but only 71.7-79.4% similarities to 17 reference sequences representing subtypes 6a-6q and to isolates km41 and gz52557. VT21, TV241 and TV249 displayed genome lengths of 9407, 9460 and 9445 nt, respectively. All three isolates contained a single open reading frame of 9051 nt while the 5'UTRs and 3'UTRs were 324-338 nt and 32-71 nt, respectively. They shared common sizes with QC227/6o and QC216/6p isolates in all ten protein regions. Phylogenetic analyses demonstrated that VT21, TV241 and TV249 clustered independently and were assigned subtype 6t, following the recent designations of 6r and 6s. Analysis of partial genomic sequences available for genotype 6 variants revealed five additional subtype 6t isolates, all originating from Vietnam. This analysis revealed two additional groups of isolates, and at least seven novel variants analogous to km41 and gz52557 that group independently and do not classify within the subtypes 6a-6t. This suggests the existence of at least 11 additional subtypes for genotype 6. In addition, the existence of isolates showing genetic distances greater than those within subtypes, but lesser than those between subtypes, raises interesting questions regarding the classification of HCV.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef.

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

Phenotypic and functional analysis of immune CD8+ T cell responses induced by a single injection of a HIV DNA vaccine in mice.

HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-gamma-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. Using IFN-gamma-ELISPOT and multiparametric FACS analysis, we characterized the induced CMI response. We found that the response was detectable for at least 63 wk. ELISPOT detection of IFN-gamma-producing T cells showed a profile with two waves separated by a long period of minimal response. Multiparametric FACS analysis showed two populations of CD3(+)CD8(+) T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-gamma. Both IFN-gamma-producing and non-IFN-gamma-producing HIV-specific CD8(+) T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Our longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-gamma-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines.