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. 2014 Aug 1;193(3):1324-32.
doi: 10.4049/jimmunol.1400222. Epub 2014 Jun 23.

Mucosal polyinosinic-polycytidylic acid improves protection elicited by replicating influenza vaccines via enhanced dendritic cell function and T cell immunity

José V Pérez-Girón  1 , Alan Belicha-Villanueva  2 , Ebrahim Hassan  1 , Sergio Gómez-Medina  1 , Jazmina L G Cruz  1 , Anja Lüdtke  1 , Paula Ruibal  1 , Randy A Albrecht  3 , Adolfo García-Sastre  4 , César Muñoz-Fontela  5
Affiliations

Mucosal polyinosinic-polycytidylic acid improves protection elicited by replicating influenza vaccines via enhanced dendritic cell function and T cell immunity

José V Pérez-Girón et al. J Immunol. .

Abstract

Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity.

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Figures

Figure 1
Figure 1. Effect of mucosal administration of poly IC on cold-adapted vaccine protection
A. Cold-adapted influenza viruses (CAV) harboring HA and NA from the PR8 strain were rescued using reverse genetics protocols. Influenza-permissive Madin-Darby canine kidney (MDCK) cells were infected with CAV at a multiplicity of infection (MOI) of 0.1 for 48h. Cells were fixed and permeabilized and influenza virus was detected by immunofluorescence using anti-NP-FITC antibodies. The white circle indicates an area of viral replication. B. Vaccine safety was assessed in C57BL6/J mice by intranasal administration of the indicated CAV doses and monitored daily for weight loss and clinical signs for up to 20 days. Results represent an average from at least three independent experiments. C. Mice were vaccinated with 103 pfu of CAV in the presence of 20 μg of poly IC or poly IC administered 24h after vaccination. Control mice received PBS instead of vaccine (MOCK-vaccinated). At day 20 post-vaccination mice were challenged with 105 pfu of PR8 (100 × LD50). Mouse weight was monitored daily and mice were sacrificed when they reached the ethical endpoint of 25% loss of their initial weight as per animal approval guidelines. D. Average body weight of mice in all vaccine groups after lethal PR8 challenge. Asterisks denote statistical significance p< 0.05 as assessed by Two-Way ANOVA followed by a Bonferroni post-test. Results represent animals pooled from three independent experiments. E. Kaplan-Meier survival curve showing percentage of mice that survived to lethal influenza challenge in all the indicated conditions. Results represent animals pooled from three independent experiments.
Figure 2
Figure 2. poly IC enhances migration of antigen-bearing DCs
A. Mice vaccinated with the indicated regimens were infused with CFSE-labeled 2× 106 OT-I T cells 3 hours after vaccination. 4 days after vaccination OT-I T cell proliferation was determined in the mLNs by analysis of CFSE dilution in the CD3+ CD4- CD8+ T cell gate. Vaccination with CAV was used as a negative control while CAV + 2 μg of endotoxin-free OVA was used as positive control. B. Mice vaccinated with CAV-SIINFEKL alone, with simultaneous poly IC treatment, or treated with poly IC 24h after vaccination were sacrificed at day 3 post-vaccination. Antigen-bearing migratory CD103+ tDCs were identified as CD103+ H-2b-SIINFEKL+ cells. The graph depicted represents the average of three biological replicates. Asterisks denote statistical significance p<0.05 as assessed by Student's t test. C. Levels of vaccine-derived NP RNA were assessed in the BAL of vaccinated mice 48 h after vaccine administration using primers specific for the A/Ann Arbor/6/60 (H2N2) virus. PCR conditions and analysis are indicated in the Materials and Methods section.
Figure 3
Figure 3. Effect of poly IC on vaccine-induced adaptive immunity
A. Kinetics of total CD8 T cells in the lungs of mice subjected to the indicated vaccine regimens after viral challenge with 103 pfu of PR8. Graphs represent total number of CD8 T cells at the indicated time points in the T cell gate. At least 5 mice per time-point are shown. B. Mice vaccinated intranasally with the indicated regimens were challenged with 103 pfu of PR8. Mice were sacrificed at days 3, 6, 10, and 25 post-infection and NP366-374 antigen specific CD8+ T cells were determined in the lung by multicolor flow cytometry. FITC-conjugated dextramers harboring the immunodominant peptide NP366-374 were utilized to detect vaccine-specific CD8 T cells. Graphs indicate the percentage of dextramer+ cells in the CD8 T cell gate (CD3+ CD4- CD8+). C. Representative plots illustrating the percentage of antigen-specific CD8 T cells in the lungs at days 3 and 25 post-challenge. D. Mice vaccinated with the indicated regimens were bled at day 20 post-vaccination. Pre-immune mice (naïve) are shown for baseline levels. Serum samples were utilized in a focus reduction neutralization assay as described in Materials and Methods. Neutralization titers are represented as the reciprocal of the last dilution at which infection was completely blocked. E. Mice were vaccinated with 103 pfu of CAV alone or with CAV+ poly IC 24 h later. Control mice received PBS instead of vaccine (MOCK-vaccinated). At day 20 post-vaccination mice were challenged with 106 pfu of X-31 virus. Mouse weight was monitored daily and mice were sacrificed when they reached the ethical endpoint of 25% loss of their initial weight. Asterisks denote statistical significance p< 0.05 as assessed by Two-Way ANOVA followed by a Bonferroni post-test.
Figure 4
Figure 4. Role of TLR3 on the adjuvant effect of poly IC
A. Wild-type or TLR-3-/- mice were vaccinated with 103 pfu alone or with poly IC administered 24 hours after vaccination. Control mice received PBS instead of vaccine (MOCK-vaccinated). At day 20 post-vaccination mice were challenged with 105 pfu of PR8. Infection control mice were infected with PR8 in the absence of vaccination. Mouse weight was monitored daily and mice were sacrificed when they reached the ethical endpoint of 25% loss of their initial weight. Results represent individual animals and average from at least three independent experiments. Asterisks denote statistical significance p< 0.05 as assessed by Two-Way ANOVA followed by a Bonferroni post-test. B. Mouse serum at the indicated dilutions was used to coat confluent MDCK cells that were later infected with PR8-GFP at an MOI of 1. Neutralization of virus infection was assessed by evaluation of GFP expression. Naïve (pre-immune) mice were also bled for evaluation of baseline antibody levels.
Figure 5
Figure 5. Cell types responsible for amplification of TLR3 signaling
A. Bone marrow chimeric mice were engineered as described in Materials and Methods. Left panel: Lang-wt (Langerin-DTR 75%: wt 25%) and Lang-TLR3-/- (Langerin-DTR 75%: TLR3-/- 25%) were treated intranasally with 50 ng of diphteria toxin (DT). Daily doses were administered starting treatment at day -2 before vaccination and until day 2 post-vaccination. Mice were vaccinated with 500 pfu of X-31 or X-31+ poly IC 24 h later. At day 20 post-vaccination NP366-374-specific CD8 T cells were determined by dextramer staining in the lungs. Left panels show representative plots, and right panel shows the average percentage of dextramer+ CD8 T cells from triplicate samples. B. Bone marrow chimeric mice reconstituted with bone marrow from wild type (WTwt) or TLR-3-/- mice (WTTLR-3-/-) were vaccinated with 3 × 103 pfu of x-31 virus or x-31+ poly IC 24 h later. At day 20 post-vaccination NP366-374-specific CD8 T cells were determined by dextramer staining in the lungs. Left panels show representative plots, and right panel shows the average percentage of dextramer+ CD8 T cells from triplicate samples. C. wt and TLR3-/- mice were vaccinated with X-31 as described above and NP-specific CD8 T cells were asessed in the lungs at day 20 post-vaccination. Left panels show representative plots, and right panel shows the average percentage of dextramer+ CD8 T cells from triplicate samples. D. WT and TLR3-/- mice were lethally irradiated and reconstituted with bone marrow cells from coisogenic CD45.1+ donor mice. Upon reconstitution mice were vaccinated with 3 × 103 pfu of x-31 virus or x-31+ poly IC 24 h later. At day 20 post-vaccination NP366-374-specific CD8 T cells were determined by dextramer staining in the lungs. Left panels show representative plots, and right panel shows the average percentage of dextramer+ CD8 T cells from triplicate samples.
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