Cell-mediated responses elicited by vaccination.

<p>HLA-A2k^b transgenic mice (n = 3 per group) were inoculated (100 µl) subcutaneously at the base of the tail on days 0 and 14 with 30 µg of VLPs alone, emulsified with an equal amount of complete freund’s adjuvant (CFA) or pre-mixed with 30 µg of E₈Pam₂Cys in saline. Splenocytes were obtained 28 days later and restimulated for 7 days in the presence of 10 µg VLPs or an irrelevant HCV-derived HLA-A2-restricted epitope not part of the VLP construct. The frequency of peptide-specific T cells producing IFN-γ was determined in an ELISPOT assay. Each bar represents the average number of IFN-γ producing T cells and standard deviation in each group after subtracting non-specific responses from corresponding samples stimulated with the irrelevant peptide.</p

Neutralisation of VLP cell entry by sera from vaccinated animals.

<p>(A) Huh7 cells (5×10⁵ cells) were incubated with FITC labelled VLPs (200 ng) in the presence of a 1∶40 dilution of anti-CD81 antibody or serum from naïve mice. Cells were then washed and analysed for fluorescence by flow cytometry. (B) Neutralisation of VLP entry was determined by pre-incubating FITC labelled VLPs (200 ng) with a 1∶5 dilution of immune sera from mice inoculated with VLPs in saline, Alhydrogel or E₈Pam₂Cys. Supernatants were clarified by centrifugation, incubated with Huh7 cells (5×10⁵) in a total volume of (500 µl) for 1 hour. Cells were then harvested and cellular fluorescence levels analysed by flow cytometry. All bar graphs represent the percentage reduction in VLP entry relative to baseline levels obtained using serum from naïve mice.</p

Dendritic cell uptake of fluorescenated VLPs.

<p>CD11c⁺ MHC Class II⁺ splenocyte-derived cultured D1 dendritic cells (2×10⁵ cells) were cultured in the presence of 5 µg FITC-labelled VLPs alone or in association with E₈Pam₂Cys (0.01 nmoles/ml). Cells were harvested after 24 hours, washed, fixed in 1% paraformaldehyde and analysed for green fluorescence by flow cytometry. Representative histograms show both surface (A) and intracellular (B) associated fluorescence derived from the analysis of a minimum of 1×10⁴ cells in each sample. To determine the level of fluorescence emanating from the cell interior, extracellular fluorescence was quenched by exposing cells to trypan blue for 1 minute prior to analysis. Bar graphs show the percentage of cells deemed to be positive for (C) whole cell or (D) intracellular associated fluorescence based on a marker that was set using untreated cells and are representative of results from one of three experiments conducted separately.</p

Dendritic cell maturation.

<p>(A) D1 dendritic cells (2×10⁵ cells) were incubated with VLPs (5 µg) alone or formulated with E₈Pam₂Cys (0.01 nmoles/ml) or Alhydrogel (5 µg) in a total volume of 1 ml. For comparative purposes within all experiments, cells were also either left untreated, exposed to LPS (5 µg/ml) or to similar amounts of each adjuvant alone. Cell surface MHC class II expression was determined after 16 hours using a PE-conjugated anti-IA/IE antibody. Cells expressing low levels of MHC Class II molecules were deemed to be immature whilst those expressing high levels were considered to be mature. Shown are representative histograms depicting cell surface MHC class II expression from one of three experiments conducted separately. MHC Class II^high expressing cells are shaded in grey. For dosing experiments, cells were also incubated with increasing amounts of (B) VLPs, (C) E₈Pam₂Cys, (D) Alhydrogel or VLPs (5 µg) formulated with increasing amounts of (E) E₈Pam₂Cys, or (F) Alhydrogel.</p

Antibody responses elicited by vaccination.

<p>Groups of BALB/c mice (n = 5 per group) were inoculated (100 µl) sub-cutaneously at the base of the tail with 20 µg of VLPs alone, pre-incubated with an equal volume of aluminium hydroxide (13 mg/ml) or 60 µg of E₈Pam₂Cys in saline on days 0 and 21. (A) VLP-specific antibody levels in sera prepared from blood taken on days 21 (white circles) and 35 (black circles) were then determined by ELISA. Individual animal antibody titres are presented with the mean value represented by the horizontal bar. An additional dose of each formulation was also administered to 3 mice from each group on day 56 and antibody titres determined on day 63 (grey circles). (B) E2-specific antibody titres in these animals were also determined at this same time point.</p

Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses

<div><p>CD8⁺ T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8⁺ T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8⁺ T cell responses and the establishment of immunological CD8⁺ T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8⁺ T cell responses. Importantly, functional memory CD8⁺ T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4⁺ T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8⁺ T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.</p></div

Statistically significant cytokines with oseltamivir-associated reductions in the nasal wash, adjusted by viral loads.

<p>Statistically significant cytokines with oseltamivir-associated reductions in the nasal wash, adjusted by viral loads.</p

Normal influenza-specific CD4⁺ T cell responses in oseltamivir-treated mice after secondary infection.

<p>Mice were treated with PBS or oseltamivir and infected with influenza virus X31, then rested for 40 days. At this time point, mice were infected i.n. with 600 pfu of influenza PR8. At 8 days post-secondary infection, (A) MedLN and (B) BAL fluid were harvested and assessed for the total number of influenza IA^b- NP₃₁₁-specific CD4⁺ T cells by intracellular cytokine staining after <i>ex vivo</i> stimulation. These data represent 2 individual experiments with 4–5 mice per group. Error bars represent standard error of the mean. n.s., not significantly different.</p

Recall CD8⁺ T cell responses are not compromised in mice with oseltamivir-interrupted primary influenza infection.

<p>(A) Naïve female BL/6 mice were administered either oseltamivir or PBS four hours prior to intranasal infection with 10⁴ pfu of HK and then once daily for eight days. Mice were secondarily challenged i.n. with 600 pfu of PR8 either (CD) 55 or (EF) 120 days after primary infection. (B) Mice were monitored daily for body weight loss. D^bNP₃₆₆ and D^bPA₂₂₄-specific CD8⁺ T cells in (CE) BAL and (DF) spleen were enumerated by intracellular staining of IFN-γ after five hours of stimulation with NP₃₆₆ or PA₂₂₄ peptide. Data represent the mean and standard deviation of a single experiment with 4–5 mice per group. Similar results were observed from two further recall experiments at day 55 after primary infection.</p

Oseltamivir treatment decreases the magnitude of the effector influenza-specific CD8⁺ T cell responses.

<p>BL/6 mice administered with oseltamivir or PBS were assessed for CD8⁺ T cell responses to the influenza A viral epitopes (A) D^bNP₃₆₆, (B) D^bPA₂₂₄, (C) K^bPB1_703-711, (D) D^bPB1-F2_62-70 and (E) K^bNS2_114-121 by intracellular IFN-γ staining after a five hour stimulation of splenocytes with cognate peptide. Data represent 9–10 mice per group, analysed over two independent experiments. Epitope-specific CD8⁺ T cells directed at (FG) D^bNP₃₆₆, (HI) D^bPA₂₂₄ were enumerated by tetramer staining in both spleen (GI) and at the site of infection (FH). Data represent 9–10 mice per group, analysed over two independent experiments. The mean and standard deviation is shown. (JK) Representative dotplots for the D^bNP₃₆₆⁺CD8⁺ and D^bPA₂₂₄⁺CD8⁺ T cell populations on d10 (acute phase) and d40 (memory) are shown; populations are gated on CD8⁺ T cells. The mean and standard deviation is shown. *P<0.05; **P<0.01.</p