Vaccinated macaques have reduced viral RNA loads and viral replication in nasal washes and lungs.

<p>Three weeks post final vaccination (week 15) control and vaccinated macaques are challenged with 10^7.4 PFU of A/California/04/2009 virus (n = 8/group). A) Bronchioalveolar lavage (BAL) viral RNA titers in control and vaccinated macaques (p<0.0079, Mann-Whitney U test). <i>In situ</i> hybridization with influenza virus specific, ³⁵S-labeled riboprobes was used to localize challenge virus RNAs in the B) right accessory and C) right caudal lung lobes of control and vaccinated macaques at day 3 post infection (n = 3/group). Viral RNA signals are evident as collections of black silver grains over cells. Animal numbers are noted in the upper portion of each micrograph.</p

DNA immunization induces influenza-specific T cell responses.

<p>A) IFN-<b>γ</b> analysis was performed on peripheral blood mononuclear cells. Vaccine-induced responses to specific influenza antigens 2 weeks post third vaccination (week 14). Mean response for each animal is plotted (n = 8/group). Error bars represent SEM. B) Responses of vaccinated animals to influenza peptide pools corresponding to subsets of HA and NP as well as M2e at 2 weeks post third vaccination (week 14). C) Total IFN-<b>γ</b> responses against HA, NP, and M2e in vaccinated and control animals at 2 weeks post third vaccination (week 14). Mean of duplicate samples is plotted; error bars represent SEM. Responses after third immunization are significantly elevated over baseline; p = 0.0008 as calculated by Mann-Whitney U test.</p

LT-MA DNA vaccine induces systemic and mucosal antibodies in NHP against HA, M2e, and NP.

<p>Cynomolgus macaques (n = 8) received 3 immunizations with the LT-MA DNA vaccine at 0, 6, and 12 weeks. Sera was collected from vaccinated macaques at various time-points and analyzed for the presence of IgG by ELISA and hemagglutinin inhibition (HI) assay. A) IgG antibody responses against the vaccine components M2e, NP and the representative HA H1N1-A/New Caledonia/20/99 (NC99) in the serum. Dashed line represents an HI titer of 1:40. B) IgG antibody responses in the bronchioalveolar lavage (BAL) collected at various time-points against the vaccine components M2e, NP and HA (NC99). C) Serum HI titers against NC99 virus. D) IC₅₀ titers of vaccinated macaque serum against the matching vaccine strain NC99 and the heterologous unmatched strains PR8 and CA09. Neutralization assays were performed on serum samples taken from vaccinated macaques two weeks after the final immunization.</p

Vaccinated macaques displayed more rapid immune response and less inflammation.

<p>A) Inflammatory cytokines were assayed by Bio-Plex using BAL obtained from vaccinated and control macaques on day 3 and day 7 following CA09 challenge (day 3 n = 8 per group, day 7 n = 5 per group). Percentage of B) CD4+ and CD8+ T cells (P = 0.018), C) B cells, and D) macrophages in BAL obtained from vaccinated and control macaques at day 3 post-challenge. *P < 0.05, Mann-Whitney U test. Refer to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189780#pone.0189780.s003" target="_blank">S2 Fig</a></b> for a gating scheme of representative samples.</p

Protection from influenza correlates with NP-specific T cell responses.

<p>Correlation between viral RNA titer (CEID₅₀ equivalents/ml) on day 3 post-infection and A) NP-specific, B) HA-specific, C) M2e- specific ELISpot IFN-<b>γ</b> T cell responses prior to challenge at 2 weeks post third vaccination (week 14) were determined by Spearman-rank correlation tests.</p

Distinct transcriptional responses following viral challenge in control and vaccinated macaques.

<p>A) Expression profiles of the union of 3973 genes found to be differentially expressed were visualized using multidimensional scaling (MDS). The MDS shows similarities between individual profiles. Differential expression cutoff was set to |Fold Change| >1.5 and a q-value < 0.05 calculated using a moderated t-test with Benjamini-Hochberg correction. Each datapoint represents an individual in either the control (left, n = 8) or vaccinated (right, n = 8) group. Distinct color denotes the timepoint at which BAL samples were collected. The Kruskal stress value denotes the quality of the 2D representation of differential gene expression between groups as a fraction of the information lost during the dimensionality reduction procedure. (B) Overlap of DE transcripts in vaccinated and control macaques across time. C) Top 10 canonical pathways found to be significantly enriched at day 3 post challenge in vaccinated and control macaques. Bar graphs represent enrichment scores as determined by the–log10 p-value as calculated by right-tailed Fisher’s exact test. D) Biological functions associated with differentially expressed genes uniquely in vaccinated macaques at day 3 post challenge. Significantly enriched biological functions were those in which |activation z-scores < 2|. Activation z-scores are denoted in by the color of the bubbles. Red indicates upregulation and blue indicates inhibition of biological functions. Bubble diameter represents the–log10 p-value. Crosses signify no observable enrichment of a biological function.</p

Protection from influenza is mediated by T cell responses and not antibody responses.

<p>A) Passive transfer in mice of purified IgG from vaccinated and control macaques followed by influenza challenge with 10 MLD₅₀ CA09 virus. Survival of BALB/c mice (n = 10 per group) passively immunized (intraperitoneally) with 25 mg IgG 24 hrs before challenge with a lethal dose of CA09 influenza virus. B) Sum of responses of vaccinated animals to influenza peptide pools corresponding to subsets of HA, NP and M2e at 3 weeks post challenge (week 18) p<0.05. Spice graphs displaying cumulative polyfunctional C) CD4+ and D) CD8+ T cell responses following stimulation with HA, NP, and M2e peptide pools at baseline (week 0), post vaccination (week 14) and 11 days post infection (DPI) (week 16+4 days). The pie charts summarize the cumulative CD4+ and CD8+ T cell responses to the various effector functions as ether mono-or poly-functional responses.</p