Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jun 13;8(6):e65964.
doi: 10.1371/journal.pone.0065964. Print 2013.

The adjuvant activity of alphavirus replicons is enhanced by incorporating the microbial molecule flagellin into the replicon

Maria L Knudsen  1 , Daniel X Johansson, Linda Kostic, Eva K L Nordström, Karin Tegerstedt, Anna Pasetto, Steven E Applequist, Karl Ljungberg, Jean-Claude Sirard, Peter Liljeström
Affiliations

The adjuvant activity of alphavirus replicons is enhanced by incorporating the microbial molecule flagellin into the replicon

Maria L Knudsen et al. PLoS One. .

Abstract

Ligands of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) stimulate innate and adaptive immune responses and are considered as potent adjuvants. Combinations of ligands might act in synergy to induce stronger and broader immune responses compared to stand-alone ligands. Alphaviruses stimulate endosomal TLRs 3, 7 and 8 as well as the cytoplasmic PRR MDA-5, resulting in induction of a strong type I interferon (IFN) response. Bacterial flagellin stimulates TLR5 and when delivered intracellularly the cytosolic PRR NLRC4, leading to secretion of proinflammatory cytokines. Both alphaviruses and flagellin have independently been shown to act as adjuvants for antigen-specific antibody responses. Here, we hypothesized that alphavirus and flagellin would act in synergy when combined. We therefore cloned the Salmonella Typhimurium flagellin (FliC) gene into an alphavirus replicon and assessed its adjuvant activity on the antibody response against co-administered antigen. In mice immunized with recombinant alphavirus, antibody responses were greatly enhanced compared to soluble FliC or control alphavirus. Both IgG1 and IgG2a/c responses were increased, indicating an enhancement of both Th1 and Th2 type responses. The adjuvant activity of FliC-expressing alphavirus was diminished but not abolished in the absence of TLR5 or type I IFN signaling, suggesting the contribution of several signaling pathways and some synergistic and redundant activity of its components. Thus, we have created a recombinant adjuvant that stimulates multiple signaling pathways of innate immunity resulting in a strong and broad antibody response.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Constructs and TLR5-specific bioactivity.
(A) Schematic illustration of VREP-FliC-WT/D3. nsP = non-structural proteins (replicase); 26S = subgenomic 26S promoter. (B) Stability of flagellin expressed from VREP. Cells were infected with VREP-FliC-D3, VREP-FliC-WT or left uninfected. Cells were then pulsed with 35S-methionine and chased for 0.5 h and 2 h. Total lysate proteins (non-immunoprecipitated) were separated by SDS-PAGE and detected by autoradiography. Distinct protein bands are visible in lysates from cells infected with VREP-FliC-D3 or VREP-FliC-WT due to translational shut-off of host proteins induced by VREP. In contrast, lysates from uninfected cells are seen as a black smear due to expression of many proteins. E2A is a translational enhancer (see Materials and Methods for description). (C) TLR5 bioassay. BHK-21 cells were infected with VREP-FliC-D3 (open circle), VREP-FliC-WT (filled circle), VREP-LacZ (X) or left uninfected. Supernatants and lysates from infected BHK-21 cells were then analyzed for their signaling through TLR5 in Caco-Rumbo cells, assayed in quadruplicates. Results are expressed as fold induction of luminescence in Caco-Rumbo cells incubated with supernatant or lysate from infected cells compared to incubation with supernatant or lysate from uninfected cells.
Figure 2
Figure 2. Antibody responses against β-Gal expressed from VREP.
129sv/ew (A) or BALB/c (B) mice were immunized with the indicated regimen. Doses used were: 106 infectious units (IU) of VREP particles, 0.2 µg sFliC-D3 and 1 µg sFliC-WT. When two different VREP particles were given to the same mouse, 5×105 IU of each VREP was given. Each immunized group consisted of five mice, and one to two control mice was used. Serum was assayed for anti-β-Gal IgG by ELISA. A one-way ANOVA with Bonferroni post-hoc test of the response between vaccinated groups revealed no significant differences.
Figure 3
Figure 3. Antibody responses induced by different doses of VREP-FliC-D3.
129sv/ew mice were immunized with β-Gal alone or with adjuvant, as indicated. Three different doses of VREP-FliC-D3 and VREP-OVA were used: 106 IU, 105 IU and 104 IU. Control mice were given PBS. Each immunized group consisted of five mice, and four control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given the same dose of adjuvant as well as between groups given adjuvant and the control group given β-Gal without adjuvant. *P<0.05, **P<0.01 and ***P<0.001.
Figure 4
Figure 4. Antibody responses after multiple immunizations.
C57/BL6 mice were immunized two times with OVA protein alone or mixed with 106 IU VREP-FliC-D3. Control mice were given PBS. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-OVA IgG by ELISA after one (black bars) or two (gray bars) immunizations. A Student’s t-test was used to compare the response after one and two administrations of the same vaccine. * P<0.05.
Figure 5
Figure 5. Antibody responses induced by VREP and sFliC-D3.
129sv/ew mice were immunized with β-Gal alone or with indicated adjuvant. VREP-OVA is indicated as ‘VREP’, and the dose used was 106 IU. Doses of sFliC-D3 are indicated in the figure. Control mice were given PBS. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given the same dose of sFliC-D3, between mice given VREP+sFliC-D3 and mice given VREP, as well as all adjuvanted groups and mice given β-Gal alone. **P<0.01 and ***P<0.001.
Figure 6
Figure 6. Adjuvant effect of VREP encoding FliC-WT or FliC-D3.
129sv/ew mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 106 IU of VREP constructs were used. Control mice were given PBS. Each immunized group consisted of five to six mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between mice given sFliC-WT and VREP-FliC-WT, between mice given sFliC-D3 and VREP-FliC-D3, between mice given VREP-FliC-WT or VREP-FliC-D3 and VREP, as well as all adjuvanted groups and mice given β-Gal alone. *P<0.05, **P<0.01 and ***P<0.001.
Figure 7
Figure 7. Contribution of type I IFN signaling.
Ifnar1 −/− (gray bars) and 129sv/ew (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 106 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five mice, and two control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2a by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Ifnar1 −/− mice given the same vaccination. *P<0.05, **P<0.01 and ***P<0.001.
Figure 8
Figure 8. Contribution of TLR5 signaling.
Tlr5 −/− (gray bars) and C57BL/6N (black bars) mice were immunized with β-Gal alone or with indicated adjuvant. 0.2 µg of soluble flagellin and 106 IU of VREP constructs were used. Control mice were given VREP-OVA. Each immunized group consisted of five (WT) or six (Tlr5 −/−) mice, and two-three control mice were used. Serum was assayed for anti-β-Gal IgG, IgG1 and IgG2c by ELISA. A one-way ANOVA with Bonferroni post-hoc test was used to compare the response between WT and Tlr5 −/− mice given the same vaccination. *P<0.05, **P<0.01 and ***P<0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Coffman RL, Sher A, Seder RA (2010) Vaccine adjuvants: putting innate immunity to work. Immunity 33: 492–503. - PMC - PubMed
    1. Pasare C, Medzhitov R (2005) Control of B-cell responses by Toll-like receptors. Nature 438: 364–368. - PubMed
    1. Querec TD, Akondy RS, Lee EK, Cao W, Nakaya HI, et al. (2009) Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol 10: 116–125. - PMC - PubMed
    1. Querec T, Bennouna S, Alkan S, Laouar Y, Gorden K, et al. (2006) Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity. J Exp Med 203: 413–424. - PMC - PubMed
    1. Kasturi SP, Skountzou I, Albrecht RA, Koutsonanos D, Hua T, et al. (2011) Programming the magnitude and persistence of antibody responses with innate immunity. Nature 470: 543–547. - PMC - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by the Swedish Research Council (www.vr.se) and the Swedish International Development Cooperation Agency (www.sida.se). MLK is a recipient of Karolinska Institutet Faculty Funding for Doctoral Students (www.ki.se). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.