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Comparative Study
. 2015;11(9):2305-11.
doi: 10.1080/21645515.2015.1047567. Epub 2015 Jun 19.

Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses

Aaron H Rose  1 , FuKun W Hoffmann, Jared H Hara, Johann Urschitz, Stefan Moisyadi, Peter R Hoffmann, Pietro Bertino
Affiliations
Comparative Study

Adjuvants may reduce in vivo transfection levels for DNA vaccination in mice leading to reduced antigen-specific CD8+ T cell responses

Aaron H Rose et al. Hum Vaccin Immunother. 2015.

Abstract

Adjuvants for DNA vaccination are designed to promote transformation of transgenes into target cells and increase inflammation in the site of injection, with resultant immune cell recruitment. Numerous studies indicated cationic liposomes as effective adjuvants for DNA vaccination due to their ability to promote in vivo transfection and innate immune system activation. Commercial reagents as Adjuplex and in vivo-JetPEI are also intended to facilitate DNA vaccination. Here, we evaluate the adjuvant properties of cationic liposomes, Adjuplex and in vivo-JetPEI compared to injection of DNA without adjuvant. In mice vaccinated with piggyBac pDNA vaccines, we assessed in vivo antigen expression, innate immune responses in draining lymph nodes, and antigen-specific T cell responses in spleens and blood. Surprisingly, vaccination with DNA in PBS emerged as the most efficient in promoting in vivo transfection and consequent antigen expression, while the addition of adjuvant reduced the amount of antigen expressed. On the other hand, we discovered higher numbers of innate immune cells and activated dendritic cells in the lymph nodes of mice injected with adjuvants than those vaccinated in PBS. The analysis of eGFP-specific immune responses revealed that all the different immunizations induced functional antigen-specific T cells in spleens, although only T cells generated by non-adjuvant vaccination and Adjuplex were identified in the blood of vaccinated mice. These results provide insight into the effects of these 3 adjuvants and may facilitate appropriate use off adjuvants by researchers using DNA vaccines in laboratory animals.

Keywords: Adjuplex; DNA vaccine; JetPEI; adjuvants; cell mediated immunity; innate immunity; liposomes; piggyBac; transposase.

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Figures

Figure 1.
Figure 1.
Experimental protocol to compare antigen expression, innate immunity, and antigen-specific T cell activation after DNA vaccination with or without adjuvants. Different groups of mice were injected with piggyBac integrating DNA plasmids formulated with each of the 3 different adjuvants. Unvaccinated and non-adjuvant DNA vaccinated mice were also used. Antigen expression was evaluated in mice after the second vaccination with pmhyGENIE-3-GL3. In other mice, innate immune responses were evaluated in inguinal lymph nodes after the 1st vaccination with pmhyGENIE-3-eGFP. In another group of mice, adaptive eGFP-specific immune responses were evaluated in spleen cells isolated 10 days after the second vaccination with pmhyGENIE-3-eGFP. Presence of eGFP-specific CD8+ T cells was also evaluated in blood of pmhyGENIE-3-eGFP vaccinated mice after the second vaccination.
Figure 2.
Figure 2.
Vaccination with pmhyGENIE-3-GL3 diluted in PBS leads to improved expression of luciferase protein when compared with liposomes, Adjuplex, and JetPEI. (A) Luciferase activity from mice s.c. injected with plasmid DNA without adjuvants (Non-adjuvant) or combined with liposomes, Adjuplex or JetPEI. Control mice were not vaccinated. Data represent mean + SEM (N = 5/group). Statistical significance was evaluated using one-way ANOVA. Bonferroni multiple comparison test was performed to identify differences between control and vaccinated mice. *P < 0.05. (B) Representative IVIS images from mice unvaccinated or s.c. injected with pmhyGENIE-3-GL3 with or without adjuvants.
Figure 3.
Figure 3.
Analysis of lymph node cells after DNA vaccination in PBS, liposomes, Adjuplex, or JetPEI. Lymph nodes were isolated 72 h after vaccination with the different adjuvants and compared with lymph nodes from unvaccinated or non-adjuvant DNA vaccinated mice. Overall number of lymph node derived cells was assessed (A) and flow cytometric analysis performed to detect percentages of different immune cell populations. Results are shown for B220+ B cells (B), Gr-1+ granulocytes (C), CD11b+ macrophages (D), CD4+CD3+ T cells (E), CD8+CD3+ T cells (F) and CD11c+ dendritic cells (G). The activation of CD11c+ dendritic cells was also evaluated by analyzing the expression of CD40+ cells (H) and CD80+ cells (I). Data are expressed as mean + SEM (N = 5/group). Means were compared using one-way ANOVA followed by the Bonferroni multiple comparison test * P ≤ 0.05. Data of CD4+and CD8+cells are expressed as percentage of these cells over the population of total CD3+ cells. Data of Gr-1+, CD11b+and CD11c+cells are instead showed as percentage of the total number of lymph node cells.
Figure 4.
Figure 4.
eGFP-specific CD8+ T cells were detected in blood of mice vaccinated with DNA in PBS (Non-adjuvant) or injected with Adjuplex. (A) Flow cytometric detection of CD8+ T cells specific for the eGFP epitope corresponding to amino acids 200–208 was performed using a fluorescent pentamer. Data are shown for blood cells collected 10 days after second vaccination and represent mean + SEM (N = 5/group). Statistical significance was evaluated using one-way ANOVA. Bonferroni multiple comparison test was performed to identify differences between groups. *P < 0.05. (B) Representative images from flow cytometry analysis of blood cells stained with eGFP 200–208 pentamers.
Figure 5.
Figure 5.
Vaccination with piggybac DNA plasmids formulated with PBS or different adjuvants generates antigen-specific CD8+ T cell responses in spleens. Data from flow cytometric detection of intracellular staining for IFNγ in CD8+ T cells after pulsing with an irrelevant peptide or with eGFP 200–208 peptide. Data represent mean + SEM (N = 5/group). Means of each group were compared using Bonferroni multiple comparison test after ANOVA analysis. *P < 0.05.
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References

    1. Lu S. Immunogenicity of DNA vaccines in humans: it takes two to tango. Hum Vaccin 2008; 4:449-52; PMID:18443427; http://dx.doi.org/10.4161/hv.4.6.6179 - DOI - PubMed
    1. Sasaki S, Takeshita F, Xin KQ, Ishii N, Okuda K. Adjuvant formulations and delivery systems for DNA vaccines. Methods 2003; 31:243-54; PMID:14511957; http://dx.doi.org/10.1016/S1046-2023(03)00140-3 - DOI - PubMed
    1. Bertino P, Urschitz J, Hoffmann FW, You BR, Rose AH, Park WH, Moisyadi S, Hoffmann PR. Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8(+) T cell responses. Vaccine 2014; 32:1670-7; PMID:24513010; http://dx.doi.org/10.1016/j.vaccine.2014.01.063 - DOI - PMC - PubMed
    1. O'Hagan DT, De Gregorio E. The path to a successful vaccine adjuvant–‘the long and winding road’. Drug Discov Today 2009; 14:541-51; PMID:19508916; http://dx.doi.org/10.1016/j.drudis.2009.02.009 - DOI - PubMed
    1. McCartney S, Vermi W, Gilfillan S, Cella M, Murphy TL, Schreiber RD, Murphy KM, Colonna M. Distinct and complementary functions of MDA5 and TLR3 in poly(I:C)-mediated activation of mouse NK cells. J Exp Med 2009; 206:2967-76; PMID:19995959; http://dx.doi.org/10.1084/jem.20091181 - DOI - PMC - PubMed

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