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. 2017 Dec 2;13(12):2763-2771.
doi: 10.1080/21645515.2017.1308988. Epub 2017 May 11.

Functionally Inactivated Dominant Viral Antigens of Human Cytomegalovirus Delivered in Replication Incompetent Adenovirus Type 6 Vectors as Vaccine Candidates

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Free PMC article

Functionally Inactivated Dominant Viral Antigens of Human Cytomegalovirus Delivered in Replication Incompetent Adenovirus Type 6 Vectors as Vaccine Candidates

Aimin Tang et al. Hum Vaccin Immunother. .
Free PMC article

Abstract

T cell immunity is critical in controlling human cytomegalovirus (HCMV) infection in transplant recipients, and T cells targeting viral immediate early proteins such as IE1, IE2 and pp65 have been speculated to be more effective against reactivation. Here we report efforts to construct replication incompetent adenovirus 6 vectors expressing these viral antigens as vaccine candidates. To reduce the potential liabilities of these viral proteins as vaccine antigens, we introduced mutations to inactivate their reported functions including their nuclear localization signals. The modifications greatly reduced their localization to the nuclei, thus limiting their interactions with cellular proteins important for cell cycle modulation and transactivation. The immunogenicity of modified pp65, IE1 and IE2 vaccines was comparable to their wild-type counterparts in mice and the immunogenicity of the modified antigens was demonstrated in non-human primates.

Keywords: T cell immunity; adenovirus vector; cytomegalovirus (CMV); non-human primate; vaccine.

Figures

Figure 1.
Figure 1.
Modification altered expression levels of IE1 and IE2. Western blot analysis of HEK293 cells transfected with DNA plasmids expressing wild-type IE1 or modified IE1 (A); wild-type IE2, IE2 with two alanine substitutions (IE2(H2A)), modified IE2 (mIE2), or modified IE2 with alanine substitutions (mIE2(H2A)) (B).
Figure 2.
Figure 2.
Expression of wild-type versus modified HCMV antigens by Ad6 vectors. Western blot analysis of Per.C6 cell lysates from Ad6-Mock, Ad6-pp65 and Ad6-mpp65 (A: lane 1, 2 and 3, respectively); Ad6-IE1 and Ad6-mIE1 (B: lane 1 and 2, respectively); Ad6-IE2 and Ad6-mIE2 (C: lane 1 and 2, respectively).
Figure 3.
Figure 3.
Subcellular localization of wild-type versus modified HCMV antigens by indirect immunofluorescent staining. MRC-5 cells infected with Ad6-pp65 (A), Ad6-mpp65 (B), Ad6-IE1 (C), Ad6-mIE1 (D), Ad6-IE2 (E) and Ad6-IE2 (F) were stained with antigen-specific antibodies and antibody specific to Sp100. Cells were also nucleus stained with DAPI. Pictures were acquired for individual staining and overlaying using confocal microscopy as described in Materials and methods.
Figure 4.
Figure 4.
Comparable immunogenicity of wild-type versus modified HCMV antigens in mice. C57BL/6 × Balb/c F1 mice (n = 10) were immunized with the indicated vaccine and dose, and the spleen cells from four mice were pooled and assessed for IFN-γ secretion in response to re-stimulation with corresponding antigen pp65 (A), IE1 (B) and IE2(C) peptide pools in ELISPOT assays. Open circles in plots represent vaccines with wild-type antigens and open triangle represent vaccines with modified antigens. DMSO at the same concentration as in peptide pools was used as negative control (dashed lines). Number of spot forming cells (SFC) on y axis was plotted against vaccine doses of viral particles (vp) on x axis.
Figure 5.
Figure 5.
Modified HCMV antigens immunogenic in nonhuman primates. T cell responses in six rhesus macaques immunized with a mixture of Ad6 vector vaccines at day 0 and wk 20. The mixture contains Ad6-mpp65, Ad6-mIE1 and Ad6-mIE2 at 1 × 1010 vp per construct. PBMCs were assessed for IFN-γ secretion pre-vaccination and at weeks 4, 8, 18 and 24 post vaccination in ELISPOT assay. PBMCs were stimulated with DMSO control (A), pp65 peptide pool (B), IE1 peptide pool (C) or IE2 peptide pool (D). IFN-γ secreting cell numbers per 1 × 106 PBMCs for each monkey were plotted in colored dashed lines and geometric mean values from all monkeys were plotted in solid black line. Number of spot forming cells (SFC) on y axis was plotted against time points of PBMC sampling.

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