Responses to multiple injections with alum alone compared to injections with alum adsorbed to proteins in mice.

New effects and mechanisms of alum on innate immunity have emerged in recent years. A number of cellular and molecular mechanisms induced by aluminum adjuvant have been reported, while the role of NALP3 and inflammasome in the cellular pathway induced by alum is still controversial. The effect of injection of alum alone without vaccine antigen into human has not been reported so far. Recently, in a phase IIIa double-blinded placebo controlled clinical trial testing the therapeutic HBsAg-anti-HBs vaccine formulated with alum against chronic viral hepatitis B patients, the placebo group receiving alum only showed substantial therapeutic effects. To explore possible underlying therapeutic mechanisms, mice were treated either with multiple injections of alum alone or with alum adsorbed to proteins (HBsAg-anti-HBs). After 4 injections Gr1(+)/CD11b(+) cells in the spleen were increased in both alum alone and alum adsorbed in proteins groups. Increased Gr1(+)/CD11b(+) cells in spleens remained consistently high in the alum alone treated group, while Gr1(+)/CD11b(+)cells decreased in the alum adsorbed to proteins group after 6 injections. Both treatments triggered increased levels of TNF-alpha measured in the plasma, but only the alum alone treated mice showed increased levels of IL-10. Histology of the liver tissues revealed a higher number of spotty necrotic foci in the alum alone immunized group. Taken together, potent inflammatory responses were induced in the alum alone immunized mice, which suggests that the substantial therapeutic effects observed in chronic hepatitis B patients immunized with alum alone might be attributed to inflammatory responses.

[Immunogenicities and comparison of DNA vaccines encoding pol genes derived from B`/C and A/E recombinant HIV-1 strains].

OBJECTIVE: To construct and compare the immunogenicities of DNA vaccines expressing pol genes derived from B`/C and A/E recombinant subtypes of HIV-1 in China. METHODS: Two DNA vaccines were constructed by inserting the codon optimized pol genes derived from B'/C and A/E subtypes of HIV-1 into mammalian expression vector pSV1.0. In vitro expression efficiencies of the two DNA vaccines were determined by Western blotting and their immunogenicities were compared by i.m. immunizing female BALB/c mice. After immunization, mice splenocytes were isolated sterilely and IFN-γ based enzyme linked immunospot assay (ELISPOT) was employed to read out the specific T cell immunity. RESULTS: The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blotting result showed both of the two DNA vaccines could be expressed at appreciable levels in vitro. Under the stimulation of Consensus B Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (636±178) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (468±265)SFCs/10(6) splenocytes (P=0.412). Under the stimulation of HIV-1 AE2f Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (1378±611) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (713±61) SFCs/10(6) splenocytes (P=0.134). Further analysis suggested pSVAE-Pol induced specific T cell responses mainly focused on Pol 1 peptide pool, while, in addition to induce Pol 1 specific T cell responses, pSVCN-Pol could also elicit T cell responses against consensus B Pol 2 peptide pool. CONCLUSION: Although pSVAE-Pol was more immunogenic, pSVCN-Pol could induce T cell responses against broader epitope spectrum. Rational vaccine design may need combine them together.

Chloramphenicol improved expression of recombinant cholera toxin B subunit in Escherichia coli and its adjuvanticity.

BACKGROUND: Cholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine. METHODS: Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl ß-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity. RESULTS: Chloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3) system and could be optimized at the concentration of 0.625 µg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group ((734 ± 240) spot forming cells/10(6) splenocytes) was higher than that induced by non-adjuvanted ((520 ± 150) spot forming cells/10(6) splenocytes), all responses against different antigens were enhanced in parallel. CONCLUSION: CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.

HIV-specific IL-2(+) and/or IFN-γ(+) CD8(+) T cell responses during chronic HIV-1 infection in former blood donors.

OBJECTIVE: Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 Tcell responses and HIV-1 viral load or CD4 count over the course of infection. In this study, 153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors. METHODS: The patients were stratified into three groups according to CD4 count: CD4≥500 cells/µL; 350 cells/µL≤CD4<500 cells/µL; CD4<350 cells/µL. PBMCs were isolated from the patients' anticoagulated blood samples. IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay. RESULTS: An overall inverse correlation were observed between CD4 count and plasma viral load. Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses, CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/µL, no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/µL to 500 cells/µL, significant correlation was only observed between the response breadth of IL-2+IFN-γ+ CD8 T cells and CD4 count; however, as for patients whose CD4 counts were more than 500 cells/µL, direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+ CD8 T cells and viral load or CD4 count. CONCLUSIONS: Universal consistent inverse correlation was only indentified between CD4 count and viral load. The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata, which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.

Establishing a Th17 based mouse model for preclinical assessment of the toxicity of candidate microbicides.

BACKGROUND: To effectively block the invasion of human immunodeficiency virus (HIV)-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because these will facilitate transepithelial viral penetration and replication. However, existing models fail to predict and evaluate vaginal mucosal toxicity induced by microbicides, and most importantly, they are unable to identify subtle or subclinical inflammatory reactions. This study was designed to develop a cost-effective in vivo model to evaluate microbicide safety in a preclinical study which can recapitulate the mucosal topical reaction. METHODS: A murine model was employed with nonoxynol-9 (N-9) as the topical stimulant within the vagina. Different concentrations of N-9 (1%, 3%, and 4%) were topically applied to the vagina for five consecutive days. A panel of inflammatory cytokines including interleukine-2 (IL-2), IL-4, IL-6, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and immuno-regulatory IL-10 were assayed in vaginal lavage. Cytokines were quantified by using cytometric bead array (CBA) and reverse transcript (RT) real-time PCR. Histopathological evaluation of vaginal tissues was conducted on hematoxylin-eosin stained slides and scored with a semi-quantitative system according to the severity of epithelial disruption, leucocyte infiltration, edema, and vascular injection. The association between the cytokines and histopathological scores was assessed by linear regression analysis. RESULTS: All three concentrations of N-9 induced inflammatory cytokine production. The 4% N-9 application resulted in a consistent production of cytokines in a time-dependent manner. The cytokines reached peak expression on day three with the exception of IL-4 which reached its peak on day one. Histopathological examination of 4% N-9 treated cervicovaginal tissues on day three showed intensive damage in four mice (sores: 10 - 13) and moderate damage in one mouse (score: 8), which were significantly associated with both inflammatory cytokines IL-17A and IL-6 and anti-inflammatory cytokines IL-4 and IL-10. Interestingly, IL-17A showed significant positive association with inflammatory cytokine TNF-α (r = 0.739; P < 0.05), anti-inflammatory cytokines IL-10 (r = 0.804; P < 0.01) and IL-4 (r = 0.668; P < 0.05). CONCLUSIONS: Our data demonstrate that a panel of cytokines (IL-17A, IL-6, IL-4 and IL-10) could be used as surrogate biomarkers to predict the histopathological damage. Th17 may play a central role in orchestrating inflammatory cytokine responses. This Th17 based mouse model is cost-effective and suitable to assess the toxicity of candidate microbicides in preclinical studies.

[Effect of GBV-C/HIV coinfection on HIV/AIDS disease progression and HIV replication].

Several research groups have recently reported that persistent GB virus C (GBV-C) co-infected with human immunodeficiency virus (HIV) leads to slower AIDSs disease progression than HIV-1 infection alone. However, these findings were not confirmed by several other studies. To investigate the association between GBV-C replication and plasma HIV loads and CD4+ T cell counts, 203 HIV-1 positive former blood/plasma donors(FBDs) were enrolled from Fuyang city of Anhui Province in China. Plasma specimens were collected from them and were tested for GBV-C using RT-PCR and ELISA. Out of 203 specimens, 52 (25.6%) cases were positive for GBV-C, including 35 male (67.3%) and 17 female (32.7%) cases. No significant association was identified between GBV-C infection and CD4+ T-cell counts or between GBV-C infection and HIV viral loads. Since all the subjects studied were naive to ART, the influence of therapy on AIDS disease progression was ruled out in this study. Overall, our data indicated that HIV-1 positive male FBDs were prone to be infected, GBV-C coinfection with HIV-1 does not significantly influence HIV/AIDS disease progression during the late stage of chronic HIV-1 infection.

[Intranasal priming with HIV DNA vaccine and systemic boosting with recombinant vaccinia induce vigorous immune responses: experiment with mice].

OBJECTIVE: To explore the strategy to raise both mucosal and systemic anti-HIV-1 immunity. METHODS: Eighteen BALB/c rats were randomly divided into 2 groups, experimental group and control group. The experimental group were further subdivided into 4 subgroups of 3 mice: 3-dose HIV DNA vaccine group, 3-dose DNA vaccine + cholera toxin (CT) adjuvant subgroup, 1-dose recombinant Tiantan strain vaccinia-based vaccine subgroup, and 3-dose DNA vaccine + CT adjuvant + Tiantan strain vaccinia-based vaccine subgroup. The control group was subdivided into 2 subgroups of 3 mice: 3-dose DNA blank vector subgroup, and 3-dose DNA blank vector + Tiantan strain vaccinia-based vaccine subgroup. Intranasal administration of DNA vaccine-based vaccine (10 microg) was done on the days 0, 14, and 28 as the mucosal priming, and recombinant Tiantan vaccinia (1 x 10(7) PFU) was injected intramuscularly as systemic boosting on the day 42. On the day 56 the mice were killed and specimens of serum, nasopharynx wash, lung wash, and spleen were collected and splenocytes were isolated. Splenocytes were added into the phosphate-buffered saline with anti-mouse interferon-gamma (IFN-gamma) envelop antibody to count the number of spot-forming cells (SFCs). Indirect ELISA was used to detect the HIV-1 specific antibody in the nasopharynx wash and lung wash. Immunohistochemistry was used to detect the intracellular staining of IFN-gamma in the splenocytes. RESULTS: The number of spot forming cells in the HIV-1 DNA vaccine + CT adjuvant group was (14 +/- 11) SFCs/10(6) splenocytes, significantly more than that of the HIV-1 DNA vaccine group [(2 +/- 1) SFCs/10(6) spleen cells (P < 0.01). The number of SFCs of the 1-dose DNA-vaccine subgroup was [(30 +/- 18) SFCs/10(6) spleen cells], significantly higher than that of the only DNS vaccine group (P < 0.01). The number of SFCs of DNA vaccine + CT adjuvant + recombinant Tiantan vaccinia-based vaccine was (61 +/- 35) SFCs/10(6) splenocytes, significantly higher than those of the other groups (all P < 0.01). Flow cytometry showed that the rate of HIV-1 Gag specific CD8(+) T cell was 1.8% +/- 1.4%. The value of specific IgG of the DNA vaccine + adjuvant + Tiantan vaccinia-based vaccine was 1.50 +/- 0.30, significantly higher than those of the blank vector, single-dose Tiantan vaccinia-based vaccine, and single-dose DNA vaccine + CT adjuvant subgroups (0.42 +/- 0.02, 0.74 +/- 0.13, and 0.75 +/- 0.02 respectively, all P < 0.05). In different subgroups the levels of specific IgA in the lung wash were all higher than those in the nasopharynx wash. The levels of specific IgA in the lung and nasopharynx wash of the DNA vaccine + CT adjuvant subgroup were higher than those of the other subgroups whether or not with boosting of Tiantan. The specific IgA levels of the groups enhanced by Tiantan vaccinia-based vaccine were all significantly higher than those of the corresponding subgroups without enhancement (all P < 0.01). The IgA level of lung wash of the DNA vaccine + CT adjuvant subgroup was 1.82 +/- 0.76, significantly higher than that of the one-dose Tiantan vaccinia-based vaccine group (0.52 +/- 0.19, P < 0.05). CONCLUSION: The vaccination modality of mucosal priming and systemic boosting induces both mucosal and systemic immune responses.

[Capacity of HIV enzyme immunoassay diagnostic kits to detect antibodies against different genotypes of HIV].

OBJECTIVE: To investigate the capacity of commercial HIV enzyme immunoassay (EIA) diagnostic kits to detect antibodies against different genotypes of HIV. METHODS: HIV RNA was detected with RT-PCR from samples positive for HIV antibody. The purified PCR products were sequenced directly and the genotypes of HIV from samples were analyzed. The samples for each genotype of HIV were diluted and the diluted samples were detected with different HIV EIA diagnostic kits. RESULTS: All 20 samples positive for HIV antibody were also positive for HIV RNA; 9 of 20 isolates were genotype B, 9 of them were genotype C or CRF BC, 2 of them were CRF AE. The sensitivity of different HIV EIA diagnostic kits to detect antibodies against different genotypes of HIV was not significantly different. CONCLUSION: The capacity of commercial HIV diagnostic kits to detect antibodies against different HIV genotypes may not be significantly different.