Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant

<div><p>Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of <i>Salmonella typhimurium</i> (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized <i>M</i>. <i>tuberculosis</i>. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.</p></div

DNA and Prime-Boost Immunizations.

<p>DNA and Prime-Boost Immunizations.</p

Intranasal delivery of Ad-encoded flagellin caused lung pathology.

<p>Mice were primed twice with DNA vaccine via the IM route and boosted IN with Ad vaccine 3 weeks later as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>. At days 1 and 10 post-boosting, mice were euthanized, lungs were harvested from different vaccine groups and lung sections were prepared for H&E staining and histopathological evaluation. Representative lung sections from mice in different vaccine groups are shown at 100x and 200x magnification. Representative data from one of two independent experiments are shown (n = 3).</p

Boosting with Ad-encoded flagellin via intramuscular or intranasal routes caused transient morbidity in mice.

<p>Mice were primed twice with DNA vaccine via the IM route, and some groups were boosted either IM or IN with Ad vaccine 3 weeks later as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>, Body mass was monitored post-systemic (A) or mucosal (B) boosting with Ad vaccines, or post-DNA priming (C) Data shown represent percentage of average change in body mass ± SEM per group for 5 individual mice within each group (*<i>p</i> < 0.05 vs 85B/85B group). Representative data from one of two independent experiments are shown (n = 5).</p

Enhancement of pulmonary mucosal and circulating Ag85B-specific CD4+ and CD8+ T cell responses when flagellin was co-expressed in the priming phase of mucosal prime-boost immunization.

<p>Mice were primed twice with DNA vaccine via the IM route and boosted IN with Ad vaccine encoding Ag85B and/or flagellin 3 weeks later as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>. At week 3 post-boosting, CD4+ and CD8+ T cell responses in spleen (A&D respectively), lung-associated lymph nodes (B&E respectively), and peripheral lung tissues (C&F respectively),were assayed by IFN-γ ELISpot. Data shown are mean of SFCs ± SEM, (*<i>p</i> < 0.05; **<i>p</i> < 0.01 vs 85B/85B group). Representative data from one of three independent experiments are shown (n = 5).</p

Ad-encoded flagellin increased levels of inflammatory factors in the circulation and pulmonary tissues.

<p>Mice were primed twice with DNA vaccine via the IM route and boosted either IM or IN with Ad vaccine 3 weeks later as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>. Levels of inflammatory cytokines and chemokines in serum samples following systemic (A) or mucosal (B) boosting, and in BALF (C)post-mucosal boosting were assayed by multiplex as described in Materials and Methods. Samples were collected from mice at 24 hours post-boosting. Cytokine concentrations are depicted as means ± SEM in each group (*<i>p</i> < 0.05; **<i>p</i> < 0.01 vs 85B/85B group). Representative data from one of two independent experiments are shown (n = 4).</p

Co-expression of flagellin at the priming phase of immunization increased numbers of splenic and mucosal Ag85B-specific CD4+ and CD8+ T cells.

<p>Mice were primed twice via the IM route with DNA vaccines encoding either Ag85B or flagellin, or both, and boosted IM with Ad vaccine vectors encoding Ag85B or flagellin, or with a cocktail of both, three weeks later as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>. At week 3 post-boosting, CD4+ T and CD8+ T cell responses in spleen (A&C respectively) and lung (B&D respectively) were assayed by IFN-γ ELISpot. Data shown are mean counts of SFCs ± SEM, (*<i>p</i> < 0.05; **<i>p</i> < 0.01 vs 85B/85B group). Representative data from one of three independent experiments are shown (n = 5).</p

Flagellin enhanced anti-Ag85B antibody responses following either systemic or mucosal prime-boost immunization.

<p>Mice were primed twice with DNA vaccine via the IM route and boosted either IM or IN with Ad vaccine three weeks later as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>. Sera were collected and tested by ELISA for anti-Ag85B IgG antibodies at week 3 post systemic (A) or mucosal (B) boosting. Data shown are means of endpoint antibody titers ± SEM per group. (C) Lungs were harvested from IN-boosted mice at week 14 post-boosting and tested for mucosal IgA-secreting B cells by ELISpot. Data shown are mean of SFCs ± SEM per group. (*<i>p</i> < 0.05; **<i>p</i> < 0.01 vs 85B/85B group). Representative data from one of two independent experiments are shown (n = 5).</p

Co-priming with flagellin enhanced Ag85B-specific CD4+ and CD8+ T cell responses following DNA immunization.

<p>Mice were immunized twice via the IM route with DNA vaccine encoding Ag85B and/or flagellin as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148701#pone.0148701.t001" target="_blank">Table 1</a>. At week 3 post-immunization, CD4+ T cell responses in spleen (A) and lung (B) were assayed by IFN-γ ELISpot. Data shown are mean of SFCs ± SEM per group, (* <i>p</i> < 0.05; **<i>p</i> < 0.01 vs DNA-85B group). CD8+ T cell responses were assayed in spleen and lungs by tetramer analysis and frequencies of Ag85B-specific tetramer positive cells within the total CD3+CD8+ parent cell population are shown (C). Intracellular cytokine staining for memory CD4+ (D) and CD8+ (E),T cells from spleen was also performed. Data shown are mean counts of Ag85B-specific cytokine-producing CD44+ memory cells per million CD3+CD4+ or CD3+CD8+ parent cells ± SEM (*<i>p</i> < 0.05 vs 85B/85B group). Representative data from one of two independent experiments are shown (n = 5).</p