DNA immunization with HA, HA+NP, and HA+NP+M2 induces similar protection after A/Vietnam/1203/2004 virus challenge in mice.

<p>(A) Survival data is shown as a percentage comparing the final animal number at day 21 with the initial animal number in each group. All HA-containing groups showed significant survival compared to controls. There is no statistical difference between the HA, HA+NP and HA+NP+M2 groups (<i>p</i> = 0.317 between HA and HA+NP; <i>p</i> = 0.146 between HA and HA+NP+M2; <i>p</i> = 0.515 between HA+NP and HA+NP+M2 by log-rank test); NP and the control groups were not statistically different from each other. (B) Body weights of the mice were also monitored and the total body weight of all of the surviving animals in each group was compared with the respective initial body weights. As expected, the HA group showed the least amount of body weight loss, with the other HA-containing groups showing similar patterns. However, the NP-immunized group demonstrated severe weight loss, similar to controls.</p

Neutralizing Antibody Responses of HA-Vaccinated Mice and Ferrets.

<p>*UD = Undetectable; NA = Not Assessed.</p><p>Neutralization was determined by lentiviral inhibition assay, hemagglutinin inhibition assay, and microneutralization assay. Sera from the indicated mouse and ferret immunizations with the indicated viral antigens by DNA alone or DNA/rAd5 before the viral challenge were evaluated by pseudotyped lentiviral inhibition, hemagglutinin inhibition (HAI), and microneutralization assays (MN). UD represents serum samples with undetectable neutralization activities even at the lowest dilutions, while NA represents samples that were not available, and therefore not assessed. In both mice and ferrets, only HA-containing groups stimulated strong humoral responses.</p

Protection of DNA/rAd5 vaccines encoding HA or HA+NP+M2, but not NP, NP+M2, or M2, against A/Vietnam/1203/2004 virus challenge.

<p>(A) Ferrets immunized three times with DNA followed by a single rAd5 boost were challenged under anesthesia with 10⁷ EID₅₀/ferret of influenza virus A/Vietnam/1203/2004. The animals were monitored 7 days for survival, shown as a percentage comparing the initial animal number to the final animal number in the same group (left panel). There was no statistical difference between the control group and groups immunized with NP, NP+M2, or M2. Both the HA and HA+NP+M2 groups showed 100% survival (right panel), whereas the vector control group showed 0% survival after the viral challenge. There was no statistically significant difference between the HA and HA+NP+M2 groups (<i>p</i> = 1.00), but there was a significant difference between these groups and the control (<i>p</i> = 0.008), by a log-rank test. (B) Body weights of the ferrets were also monitored and the total body weight of all of the surviving animals in each group was compared with the respective initial body weight (left panel). Ferrets immunized with HA and HA+NP+M2 groups showed no weight loss, while the control group ferrets showed rapid weight loss (right panel). The survival and initial animal numbers in each group on the last day of body weight data collection are indicated next to the curve labels. The survival percentage for each group was analyzed statistically by a log-rank test.</p

Expression of immunogens using DNA and rAd5 vectors in cell culture.

<p>(A) Western blot analysis confirmed the expression of HA protein of A/Thailand/1/KAN-1/2004 (lane 2), NP protein of A/PR/8/34 (lane 3) and A/Thailand/1/KAN-1/2004 (lane 4), and M2 protein of A/Thailand/1/KAN-1/2004 (lane 6) in 293T cells transfected by eukaryotic plasmid expression vectors. (B) Expression of rAd5 vectors was confirmed in A549 cells after transduction with vectors encoding HA (KAN-1) (lane 8), NP (KAN-1) (lane 9), and M2 (KAN-1) (lane 11). Arrows indicate the relevant predicted size of the indicated viral proteins. Bands refer to the right predicted size of different viral proteins that were detected in each lane as indicated. Molecular weight markers were used for protein size reference.</p

Humoral immune responses to HA, NP and M2 confirmed by ELISA after DNA/rAd5 immunization in ferrets.

<p>(A) Sera from the HA, HA+NP+M2 or vector control immunized ferrets were collected 14 days after the third DNA immunization (white bars), and 14 days after the recombinant adenovirus boost (solid bars), and subjected to ELISA assays to determine their end-point titer levels against HA(KAN-1), NP(KAN-1), and M2(KAN-1) antigens. Each bar represents the group mean for the end-point titers of total IgG and IgM, determined in duplicate by series dilution of ELISA assay with the error bars indicating the standard deviation. ANOVA tests were significant for only the responses against HA at the first time point, and for all three antigens after the rAd5 boost. For HA and M2, significant pairs of groups are noted on the graph. Only <i>p</i>-values less than 0.05 are indicated. * represents a <i>p</i>-value between 0.05 and 0.001, while ** indicates <0.001, and *** indicates <0.0001. As expected, the HA alone group elicited significant anti-HA immunity that increased after rAd5 HA boost (left panel) compared to controls (<i>p</i><0.0001). The HA+NP+M2 group elicited similar anti-HA ELISA antibodies, as well as significant anti-NP humoral responses (<i>p</i> = 0.0006) and anti-M2 responses (<i>p</i><0.0001) after the rAd boost (middle and right panels). (B) Sera from the NP, M2, NP+M2 or vector controls were collected 14 days after the third DNA immunization (white bars), and the sera from the same animals were also collected 14 days after the recombinant adenovirus boost (solid bars). ANOVA tests were not significant for any of the antigens at the first time point, and for NP and M2 after the rAd5 boost. For NP and M2, significant pairs of groups are noted on the graph. Only <i>p</i>-values less than 0.008 are indicated. * represents a <i>p</i>-value between 0.008 and 0.001, while ** indicates <0.001, and *** indicates <0.0001. For both NP and NP+M2 immunized groups, significant anti-NP humoral responses were observed after the rAd boost (<i>p</i><0.0001) (middle panel). Significant anti-M2 humoral responses were detected in animals immunized with NP+M2 post-rAd boost (<i>p</i> = 0.0005), but not in the M2 alone group (right panel).</p

image_1.PDF

<p>Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.</p

image_2.PDF

<p>Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.</p

Immunization with SLA Containing Adjuvants in Combination with WN-80E Induces Antibody Secreting Cells in Bone Marrow.

<p>In an independent experiment, we have examined the ability of WN-80E combined with SLA containing adjuvants to induce long lived antibody secreting cells (ASC) in bone marrow. Mice were immunized and bone marrow extracted after 21 days (n = 5/group). The number of antibody secreting cells was assessed by ELISPOT assay on plates coated with WN-80E. Differences between groups were compared by one way ANOVA. Adjuvant formulations containing SLA (SLA-Alum, SLA-SE) as well as emulsion alone induced significantly greater numbers of IgG+ ASC compared with antigen alone. Animals immunized with Alum or unformulated SLA also showed modest increases in numbers of ASC, but differences were not significant relative to antigen only.</p

ELISA Titers Following A Single Immunization with WN-80E.

<p>Serum antibody titers were determined by ELISA 21 days following a single immunization with WN-80E in combination with adjuvants. Titers of Total IgG (A), IgG1 (B) and IgG2c (C) were determined for all mice (n = 5/group). One way ANOVA was used to evaluate significant differences in antibody levels and PRNT titers between groups. Similar levels of Total IgG and IgG1 were observed in all immunized animals. Significantly elevated levels of IgG2c were detected in mice immunized with SLA-SE compared to those immunized with 10 μg of antigen alone (p<0.0001). Neutralizing antibody titers were also determined by PRNT assay (D) to assess antibody function. There is a trend toward increased titer in SLA-SE immunized animals at all anitgen doses, and significant increases in PRNT titer are observed at the 1μg antigen dose.</p

Immunization with SLA Containing Adjuvants in Combination with WN-80E Enhances Survival and Reduces Viral Titer to Undetectable Levels.

<p>Following a single immunization of WN-80E in combination with the indicated adjuvants, mice (n = 10/group) were challenged with 100 LD₅₀ of WNV via the intraperitoneal route. Surivial of mice was monitored over 14 days following challenge (A,B). Survival curves were compared using a Mantel-Cox test, with p-values compared to immunization with WN-80E shown. Three days post-challenge, serum was collected from all animals in order to assess virus titers. Animals immunized with SLA-Alumhad undetectable titers in all animals (P<0.0005). Those imminzed with SE or SLA-SE had minimal titers while those immunized with Alum, SLA-AF or no adjuvant showed only slightly reduced titers compared to unimmunized controls (P<0.05).</p