Single-dose protection against Plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron A - PubMed (original) (raw)

Comparative Study

. 2008 Apr;82(8):3822-33.

doi: 10.1128/JVI.02568-07. Epub 2008 Feb 6.

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Comparative Study

Single-dose protection against Plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron A

S Sridhar et al. J Virol. 2008 Apr.

Abstract

Human adenovirus serotype 5 (AdH5) vector vaccines elicit strong immune responses to the encoded antigen and have been used in various disease models. We designed AdH5 vectors expressing antigen under the control of a human cytomegalovirus (HCMV) immediate-early promoter containing its intron A sequence. The transcriptional levels of antigen and immune responses to antigen for vectors with the HCMV promoter with the intron A sequence (LP) were greater than those for AdH5 vectors using the HCMV promoter sequence without intron A (SP). We compared an E1E3-deleted AdH5 adenoviral vector, which affords more space for insertion of foreign sequences, and showed it to be as immunogenic as an E1-deleted AdH5 vector. Neutralizing antibodies to AdH5 limit the efficacy of vaccines based on the AdH5 serotype, and simian adenoviral vectors offer an attractive option to overcome this problem. We constructed E1E3-deleted human and simian adenoviral vectors encoding the pre-erythrocytic-stage malarial antigen Plasmodium berghei circumsporozoite protein. We compared the immunogenicity and efficacy of AdC6, a recombinant simian adenovirus serotype 6 vector, in a murine malaria model to those of AdH5 and the poxviral vectors MVA and FP9. AdC6 induced sterile protection from a single dose in 90% of mice, in contrast to AdH5 (25%) and poxviral vectors MVA and FP9 (0%). Adenoviral vectors maintained potent CD8(+) T-cell responses for a longer period after immunization than did poxviral vectors and mainly induced an effector memory phenotype of cells. Significantly, AdC6 was able to maintain protection in the presence of preexisting immunity to AdH5.

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Figures

FIG. 1.

FIG. 1.

Comparison of different promoters. An AdH5 adenoviral vector was constructed to express antigens MSP-142 and 85A under the control of HCMV promoters either with or without an exon A and intron A sequence. The transcriptional activity and immunogenicity of each construct were investigated and compared. (a) Schematic diagram of the promoter elements used in the adenoviral vectors. SP is an HCMV promoter in pAd/CMV/V5/DEST containing the enhancer/promoter sequence and a short regulatory sequence, while LP is the HCMV promoter containing the enhancer/promoter sequence along with the exon A and intron A sequence. (b and c) Comparison of mRNA expression levels between promoters. 293 cells were infected at an MOI of 50 with AdH5.85A.SP, AdH5.85A.LP, AdH5.MSP-142.SP or AdH5.MSP-142.LP, and RNAs were extracted from the cells 48 h after infection. Real-time RT-PCR was undertaken to compare mRNA levels of antigen transcribed by the LP or SP. Antigen mRNA levels were normalized to AdE4orf1 levels and expressed as ratios of antigen levels to AdE4orf1 levels. Panels b and c show the data for the MSP-142 and 85A antigens, respectively. Statistical analysis was performed using an unpaired t test (GraphPad Prism). **, P < 0.01. The results shown are representative of two independent experiments. (d to f) Effect of promoter on immunogenicity of adenoviral vectors. Female BALB/c mice (three/group) were immunized with 1010 vp of AdH5.85A.SP or AdH5.85A.LP (e), 1010 vp of AdH5.MSP-142.SP or AdH5.MSP-142.LP (d), or 109 vp of AdH5.85A.SP or AdH5.85A.LP (f) intradermally (i.d.) in the ear. Fourteen days later, splenocytes were assayed for IFN-γ secretion by ex vivo ELISPOT assay. Columns represent mean numbers of IFN-γ-producing SFCs per million splenocytes plus standard errors of the means (SEM). Statistical analysis was performed using a nonparametric two-way ANOVA (GraphPad Prism). *, P < 0.05; **, P < 0.01 (compared to mice immunized with vectors containing SP).

FIG. 2.

FIG. 2.

Comparison of E1-deleted and E1E3-deleted adenoviral vectors. BALB/c mice (three/group) were immunized with 1010 vp of E1-deleted AdH5.85A.LP or E1E3-deleted AdH5.85A.LP i.d. in the ear. Fourteen days later, splenocytes were assayed for IFN-γ secretion by ex vivo ELISPOT assay. Columns represent mean numbers of IFN-γ-producing SFCs per million splenocytes plus SEM.

FIG. 3.

FIG. 3.

Immunogenicity and effector T-cell development following immunization with viral vectors expressing PbCSP. (a) Immunogenicity and immune kinetics. Female BALB/c mice (three mice/group) were immunized with AdH5.PbCSP (1010 vp), AdC6.PbCSP (1010 vp), MVA.PbCSP (107 PFU), or FP9.PbCSP (107 PFU) i.d. Spleens were harvested at different time points postimmunization, and antigen-specific responses were assayed by intracellular cytokine staining for IFN-γ (CD8+ T cells) following stimulation of splenocytes with Pb9. Data are means plus SEM for each group. (b) Phenotyping of effector cell population. Data show the percentages of CD8+ IFN-γ+ CD43high cells. Columns represent means plus SEM for each group. (c) Cytolytic function of antigen-specific cells. CD8+ IFN-γ+ GrB+ coexpression is shown for representative mice at the indicated days postprime. The histograms show GrB expression (white background) after staining with anti-human GrB compared to staining with an isotype control (gray background). The number corresponds to the mean fluorescence intensity of the positive sample (black solid line).

FIG. 4.

FIG. 4.

Phenotyping of memory CD8+ T cells following viral vector immunization. Mice were immunized as described in the legend to Fig. 2. (a) Long-term evolution of CD62L and CD127 on antigen-specific cells following immunization. Splenocytes were costained for CD8, IFN-γ, CD62L, and CD127. Representative data are shown for three mice per group. (b) Development of IL-2-producing cells. Data show the percentages of CD8+ IFN-γ+ IL-2+ cells. Columns represent means plus SEM for each group. (c) CD27 expression. Data show the percentages of CD8+ IFN-γ+ CD27+ cells. Columns represent means plus SEM for each group.

FIG. 5.

FIG. 5.

Immunogenicity and efficacy of adenoviral vectors in the presence of preexisting immunity. Preexisting immunity was induced by immunizing groups of six BALB/c mice with 5 × 109 vp of AdH5.85A or C6.85A. Six weeks later, mice were immunized with adenoviral vectors expressing PbCSP at a dose of 1010 vp per mouse. (a) Effect of preexisting immunity on immunogenicity of viral vectors. Twelve days after immunization, blood was collected to analyze IFN-γ secretion by ex vivo ELISPOT assay. Data shown represent individual mouse responses, expressed in IFN-γ-producing SFCs per million peripheral blood mononuclear cells, and the line represents the mean response per group. Statistical analysis was performed using nonparametric two-way ANOVA, with adjustment for multiple comparisons (GraphPad Prism). Statistical differences are indicated as follows: **, P < 0.05; ***, P < 0.001. (b) Protective efficacy in the presence of preexisting immunity. Fourteen days after immunization with adenoviral vectors coding for PbCSP, mice were challenged intravenously with 1,000 P. berghei sporozoites. The bars represent the percentages of mice that were protected in each group. All naïve mice were infected by day 6.

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