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. 2015 Jan 15;194(2):630-6.
doi: 10.4049/jimmunol.1402530. Epub 2014 Dec 8.

Identification of Aim2 as a sensor for DNA vaccines

John J Suschak  1 , Shixia Wang  1 , Katherine A Fitzgerald  2 , Shan Lu  3
Affiliations

Identification of Aim2 as a sensor for DNA vaccines

John J Suschak et al. J Immunol. .

Abstract

Recent human study data have re-established the value of DNA vaccines, especially in priming high-level Ag-specific Ab responses, but also raised questions about the mechanisms responsible for such effects. Whereas previous reports have shown involvement of downstream signaling molecules in the innate immune system, the current study investigated the role of absent in melanoma 2 (Aim2) as a sensor for DNA vaccines. The Aim2 inflammasome directs maturation of the proinflammatory cytokines IL-1β and IL-18 and an inflammatory form of cell death called pyroptosis. Both the humoral and cellular Ag-specific adaptive responses were significantly reduced in Aim2-deficient mice in an IL-1β/IL-18-independent manner after DNA vaccination. Surprisingly, Aim2-deficient mice also exhibited significantly lower levels of IFN-α/β at the site of injection. These results indicate a previously unreported link between DNA vaccine-induced pyroptotic cell death and vaccine immunogenicity that is instrumental in shaping the Ag-specific immune response to DNA vaccines.

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Figures

Figure 1
Figure 1. Plasmid DNA vaccination induces the inflammasome
Inflammasome activation at the site of immunization was quantified by Nanostring nCounter analysis 12 hours post DNA immunization. The site of injection was harvested and mRNA was isolated and expression levels were quantified for (A) Aim2, (B) caspase-1, (C) IL-1α, (D) IL-1β. Data are the averages ± SEM of 5 mice per group.
Figure 2
Figure 2. Aim2 is required for IL-1β production in response to DNA vaccines
LPS (200 ng/ml) primed Aim2+/+ and Aim2−/− BMDMs were transfected with poly(dA-dT) or DNA vaccine plasmid for 18 hrs. (A) Secreted IL-1β in the culture supernatants was analyzed by ELISA. (B) Aim2+/+ and Aim2−/− BMDM were treated as above with the addition of the pan-caspase inhibitor Z-VAD-FMK and secreted IL-1β was quantified by ELISA. (C) Aim2+/+ and Aim2−/− BMDM were treated as above, and culture LDH amounts were reported as a percentage of lysed cellular controls. Data are presented as mean ± SEM from 3 independent experiments.
Figure 3
Figure 3. Optimal DNA vaccine immunogenicity requires Aim2
Wild-type Aim2+/+ and Aim2−/− mice were immunized intramuscularly with a pH1HA encoding DNA vaccine at weeks 2 and 4. (A) HA-specific IgG titers were analyzed fourteen days post second immunization. Anti-HA binding avidity was quantified via ELISA and reported as molar concentration of sodium thiocyanate required to displace anti-HA serum antibodies to 2x pre-bleed levels (B). For ELISPOT, spleens were harvested at termination 7 days following a 3rd boosting immunization. HA-specific antibody secreting B cells (C) or IFN-γ secreting T cells (D) in mice immunized with either pH1HA or empty vector. Data are the averages ± SEM of 5 mice per group.
Figure 4
Figure 4. Aim2-deficient mice exhibit diminished caspase-1 activation at the site of immunization
(B) Wild-type Aim2+/+ and (C) Aim2−/− mice were immunized intramuscularly with pH1HA vaccine and caspase-1 activation was quantified by FAM/FLICA staining 12 hours post immunization. (A) PBS injected controls were utilized for comparison. The site of injection was harvested and cryopreserved for tissue sectioning. FAM/FLICA staining was visualized by confocal microscopy and is representative of 3 mice per group.
Figure 5
Figure 5. Contribution of hematopoietic and non-hematopoietic cells to DNA vaccine induced immunogenicity
Bone marrow chimeric mice were immunized with a pH1HA vaccine as described in Figure 2. Fourteen days post second immunization, sera from chimeric mice were analyzed for HA-specific IgG titers (A). Spleens were harvested at termination 7 days following the 3rd immunization. Frequency of HA-specific B cells (B) or IFN-γ+ T cells (C) were reported as spots per million splenocytes in mice immunized with pH1HA vaccine. Data are the averages ± SEM of 5 mice per group.
Figure 6
Figure 6. Aim2-deficiency limits IFN-αβ production at the site of injection
Aim2 +/+ and Aim2−/− mice were immunized intramuscularly with pH1HA vaccine, and the site of injection was harvested 12 hours later. Total RNA was isolated from tissue biopsies and subjected to rt-PCR for (A) IFN-α, (B) IFN-β, (C) IP10, and (D) TNF. Reported expression levels are relative to naïve Aim2 +/+ expression. Data are the averages ± SEM of 5 mice per group.
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