Fraction of predicted N-linked glycosylation sequons in HA and NA of H3N2 and H1N1 viruses sampled in Denmark seasons 1999 to 2006

Sites with predicted potential threshold values above 0.5 are shown. Sites not shown for H3 (n = 204): 122, N2 (n = 166): 200, 329, 402, H1 (n = 27): 10, position 539 is positively predicted; however this site is located at the cytosolic region of HA and is therefore not glycosylated, N1 (= 30): 455.<p><b>Copyright information:</b></p><p>Taken from "The evolution of human influenza A viruses from 1999 to 2006: A complete genome study"</p><p>http://www.virologyj.com/content/5/1/40</p><p>Virology Journal 2008;5():40-40.</p><p>Published online 7 Mar 2008</p><p>PMCID:PMC2311284.</p><p></p

Relative prevalence of sentinel and routine influenza A viruses in Denmark 1999 to 2006

The actual numbers of influenza A positive samples for the respective seasons are as follows; 1999–2000 49, 2000–2001 28, 2001–2002 80, 2002–2003 61, 2003–2004 83, 2004–2005 91 and 2005–2006 54.<p><b>Copyright information:</b></p><p>Taken from "The evolution of human influenza A viruses from 1999 to 2006: A complete genome study"</p><p>http://www.virologyj.com/content/5/1/40</p><p>Virology Journal 2008;5():40-40.</p><p>Published online 7 Mar 2008</p><p>PMCID:PMC2311284.</p><p></p

(A) Seasonal amino acid distances of H3N2 HA and NA proteins since 1999 and (B) amino acid distances of H3N2 HA and NA from one season to the next

The same trends were observed for nucleotide distances. In 2000–2001 H1N1 viruses only were observed and therefore not included. Distance means were computed as the arithmetic mean of all pair wise distances between two seasons in the inter-season comparisons by the MEGA v.3.1 software [68]<p><b>Copyright information:</b></p><p>Taken from "The evolution of human influenza A viruses from 1999 to 2006: A complete genome study"</p><p>http://www.virologyj.com/content/5/1/40</p><p>Virology Journal 2008;5():40-40.</p><p>Published online 7 Mar 2008</p><p>PMCID:PMC2311284.</p><p></p

Cellular immune responses are induced to a similar level after immunization with IFA and CAF01.

<p>IFN-γ ELISPOT responses against (A) HLA-A2-restricted CD8 T cell epitope Vif₁₀₁ and (B) PADRE Th epitope of splenocytes from HLA-A*0201 transgenic HHD mice after 10 days of s.c. immunization with Vif₁₀₁ and PADRE in either IFA (grey bars, n = 5) or CAF01 adjuvants (black bars, n = 5) and 5 days <i>in vitro</i> prestimulation with individual epitopes. The background of an unimmunized mouse is substracted. Specific ⁵¹Cr-release from target cells preloaded with Vif₁₀₁ after incubation with effector splenocytes from Vif₁₀₁ and PADRE immunized mice adjuvanted with (C) IFA (grey, n = 5) or (D) CAF01 (black, n = 5). The percentage of specific lysis was calculated as 100×(experimental release-spontaneous release)/(total release-spontaneous release). Background lysis from an unimmunized mouse is shown (open circles). Significant positive levels are considered for >10% lysis at a 50∶1 ratio of effector:target (E:T) cells. One representative experiment out of three. SFU, spot forming units.</p

Proliferative CD8⁺ T cell responses induced after immunization with IFA and CAF01.

<p>The percentage of proliferating CD3+CD8⁺ splenocytes (having reduced CFSE dye) derived from HLA-A*0201 transgenic HHD mice 10 days after s.c. immunization with a peptide mix consisting of 10 peptides adjuvanted with (A) IFA or (B) CAF01. The background proliferation of non-stimulated (NS) cells, incubated with media alone is shown. The epitope peptides were (from left to right): NS, Gag₁₅₀, Gag₄₃₃, Env₆₇, Pol₆₀₆, Vpu₆₆, Vif₁₀₁, Vif₂₃, Gag₂₉₈ (Th), Env₅₇₀ (Th) and PADRE (Th).</p

Cellular immune responses induced after immunization in CAF01 with or without a T helper epitope.

<p>IFN-γ ELISPOT responses against (A) Vif₁₀₁ CTL epitope and (B) PADRE Th epitope of splenocytes from HLA-A*0201 transgenic HHD mice after 10 days of s.c. immunization with Vif₁₀₁ and PADRE (grey bars, n = 5) or with Vif₁₀₁ alone (black bars, n = 5) and 5 days <i>in vitro</i> prestimulation with individual epitopes. The background of an unimmunized mouse is substracted. Specific ⁵¹Cr-release from target cells preloaded with Vif₁₀₁ after incubation with effector splenocytes from individual mice immunized with (C) Vif₁₀₁ and PADRE (grey, n = 5) or (D) with Vif₁₀₁ alone (black, n = 5). The percentage of specific lysis was calculated as 100×(experimental release-spontaneous release)/(total release-spontaneous release). Background lysis from an unimmunized mouse is shown (open circles). Significant levels are considered >10% lysis at a 50∶1 ratio of effector:target (E:T) cells. One representative experiment out of three. SFU, spot forming units.</p

Identification of CD4 and CD8 T-cell epitopes within responding 15-mer peptides.

1<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-ErupLarsen1" target="_blank">[27]</a>,</p>2<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-1" target="_blank">[28]</a>,</p>3<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Nielsen1" target="_blank">[26]</a>,</p>4<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Harcourt1" target="_blank">[48]</a>,</p>5<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Rosenberg1" target="_blank">[49]</a>,</p>6<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Daucher1" target="_blank">[50]</a>,</p>7<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Wilson1" target="_blank">[51]</a>,</p>8<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Richmond1" target="_blank">[52]</a>,</p>9<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Brander1" target="_blank">[53]</a>,</p>10<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Kaufmann1" target="_blank">[54]</a>,</p>11<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Lazaro1" target="_blank">[55]</a>,</p>12<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Malhotra1" target="_blank">[56]</a>,</p>13<p>reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone.0063575-Koeppe1" target="_blank">[57]</a>.</p

Gag p24 formulated with CAF05 induces antigen-specific CD4 and CD8 T-cell responses in CB6F1 mice.

<p>Mice were immunized three times i.p. with 20 µg Gag p24 protein alone (open triangles) or adjuvanted in CAF01 (black circles) or CAF05 (open circles). Naïve mice were used as a negative control (black triangles). The T-cell response in spleens was determined three weeks after the last immunization by restimulation <i>in vitro</i> with either whole Gag p24 protein (A), the CD8 T-cell epitope AMQMLKETI-containing peptides 16–17 (B) or 10 µg/ml of a mix of overlapping Gag p24 peptides (C-D). T-cell responses were assessed by ELISA to determine the release of IFN-γ in supernatants after 4 days (A-B) or flow cytometry to determine the percentage of IFN-γ⁺ CD44^hi cells of the total CD4 (grey bars) or CD8 (black bars) T cell population (C) and the percentage of cells expressing both IFN- γ, TNF-a and IL-2 after 8 hours of stimulation (D). (A-D) Each data point represents the mean (n = 6) +/− SEM. Where the CAF05 adjuvant induced a statistically significantly higher response than CAF01 determined by two-way ANOVA and the Bonferroni post test, this has been indicated: *<i>p<</i>0.05, ***<i>p<</i>0.001. (E) The percentage of AMQMLKETI-specific CD44^hi cells of the total CD8 T-cell population was also determined in blood pooled within each group 1 week after the last vaccination.</p

Fragmentation of Gag p24 broadens the T-cell repertoire.

<p>HLA-A2/DR-transgenic mice were immunized three times with either the full set of overlapping peptides (OLGa, 2 µg/peptide) with or without CAF05 or protein (5 µg) in CAF05. Naïve mice where used as the negative control. 10 days after the last immunization, splenocytes from individual mice were restimulated with the indicated peptide pool (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063575#pone-0063575-t001" target="_blank">Table 1</a>) and the frequency of IFN-γ-producing cells was determined by ELISPOT. Shown are values after background subtraction. Black symbols indicate a significant response for that individual mice as determined by the DFR(x2) method. If a significant response was detected in either the protein/CAF05 or peptides/CAF05 group, statistically significant differences in SFU-levels between these two groups only were determined based on a one-tailed t test and <i>P</i>-values are indicated. NS, not significant.</p

T-cell recognition of peptides after immunization with equal molar doses of peptide sequences.

<p>HLA-A2/DR-transgenic mice were immunized three times with either the full set of overlapping peptides (OLGa, 2 µg/peptide) in CAF05 or protein (60 µg) in CAF05. Naïve mice where used as the negative control. 10 days after the last immunization, splenocytes from individual mice were restimulated with the indicated peptides and the frequency of IFN-γ-producing cells was determined by ELISPOT. Shown are values after background subtraction. Black symbols indicate a significant response for that individual mice as determined by the DFR(x2) method. If a significant response was detected in either the protein/CAF05 or peptides/CAF05 group, statistically significant differences in SFU-levels between these groups were determined based on a one-tailed t test and <i>P</i>-values are indicated. NS, not significant.</p