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. 2007 May;81(10):5257-69.
doi: 10.1128/JVI.00055-07. Epub 2007 Feb 28.

Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques

Amara Luckay  1 , Maninder K Sidhu, Rune Kjeken, Shakuntala Megati, Siew-Yen Chong, Vidia Roopchand, Dorys Garcia-Hand, Rashed Abdullah, Ralph Braun, David C Montefiori, Margherita Rosati, Barbara K Felber, George N Pavlakis, Iacob Mathiesen, Zimra R Israel, John H Eldridge, Michael A Egan
Affiliations

Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques

Amara Luckay et al. J Virol. 2007 May.

Abstract

Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log(10) increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

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Figures

FIG. 1.
FIG. 1.
HIV-1 antigen-specific CMI responses after pDNA immunization. Macaques were immunized i.m. at 0, 4, and 8 weeks with the pDNA expression vectors indicated in Table 1 in combination with plasmid rhesus IL-12. Two weeks after the final immunization, PBLs were collected and tested for HIV-1 Env-, Gag-, Pol-, Nef-, Tat-, and Vif-specific IFN-γ secretion by ELISPOT assay. Bars represent the average total HIV-specific IFN-γ ELISPOT responses (n = 6), with the standard errors of the means indicated.
FIG. 2.
FIG. 2.
Total HIV-specific CMI responses in CD4+ and CD8+ cell-depleted PBLs. The graphs show total HIV peptide pool-specific IFN-γ ELISPOT responses at week 10 in unfractionated, CD4+ or CD8+ cell-depleted PBLs from macaques immunized with pDNA vaccines plus plasmid IL-12. Responses were normalized to the precise number of CD3+ CD4+ or CD3+ CD8+ cells used in the assay, as determined by flow cytometry after bead depletion.
FIG. 3.
FIG. 3.
Serum antibody responses to monomeric HIV-16101 Env gp120 in rhesus macaques after pDNA immunization. Two weeks after the final pDNA immunization, geometric mean HIV-16101 Env gp120-specific IgG antibody titers (± standard errors) were determined by ELISA. Antibody end-point titers are reported as the highest reciprocal dilutions of sera giving OD450 readings threefold higher than those for week 0 (preimmune) samples. HIV-16101 gp120-specific antibody titers for group 4a were below the limit of detection (i.e., <1:100).
FIG. 4.
FIG. 4.
Changes in CBC parameters associated with in vivo EP. At baseline (week −2) and 2, 22, and 68 weeks after the final immunization (weeks 10, 30, and 76), macaques were screened for changes in peripheral blood cell subsets by CBC analysis. Data are reported as percent changes at (A) week 10, (B) week 30, and (C) week 76 relative to baseline (± standard errors) for macaques receiving pDNA vaccination with in vivo EP (EP; n = 6) versus macaques receiving pDNA vaccination without in vivo EP (non-EP; n = 30). *, statistically significant difference (P < 0.05) between EP and non-EP groups. WBC, white blood cells.
FIG. 5.
FIG. 5.
HIV-1 antigen-specific CMI responses following pDNA immunization, with and without in vivo EP. Macaques were immunized at 0, 4, and 8 weeks with the three-vector pDNA vaccine design 3c in combination with plasmid rhesus IL-12 by standard i.m. injection (A) or by i.m. injection followed by in vivo EP (B). At various time points, PBLs were collected and tested for HIV-1 Env-, Gag-, Pol-, Nef-, Tat-, and Vif-specific IFN-γ secretion by ELISPOT assay. Bars represent the average total HIV-specific IFN-γ ELISPOT responses (n = 6), with the standard errors of the means indicated. *, statistically significant difference (P < 0.05) between group 3c and group 3cE at the indicated time point.
FIG. 6.
FIG. 6.
Total HIV-specific CMI responses in CD4+ and CD8+ cell-depleted PBLs after pDNA immunization alone or in combination with in vivo EP. The graphs show average total HIV peptide pool-specific IFN-γ ELISPOT responses (± standard errors) at week 10 in unfractionated, CD4+ or CD8+ cell-depleted PBLs from macaques immunized with pDNA vaccines plus plasmid IL-12 alone (A) or followed by in vivo EP (B). Responses were normalized to the precise number of CD3+ CD4+ or CD3+ CD8+ cells used in the assay, as determined by flow cytometry after bead depletion. The ratio of the response seen in CD4+ cell-depleted PBLs to the response seen in CD8+ cell-depleted PBLs is indicated.
FIG. 7.
FIG. 7.
Serum HIV-16101 Env gp120- and HIV-1IIIB viral lysate-specific humoral immune responses in rhesus macaques after pDNA immunization, with and without in vivo EP. Geometric mean HIV-16101 Env gp120-specific IgG antibody titers (± standard errors) (A) and HIV-1IIIB viral lysate-specific IgG antibody titers (± standard errors) (B) were determined by ELISA. Antibody end-point titers are reported as the highest reciprocal dilution of sera giving OD450 readings threefold higher than those for week 0 (preimmune) samples. Arrows indicate the timing of pDNA immunization.
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