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

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

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