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

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

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

Co-expression of PD-1, 2B4 and CD160 is independent of when ART is initiated.

<p>Co-expression of PD-1 and 2B4 or of PD-1, 2B4 and CD160 was tested in ART-naïve, ART-treated and HIV-negative individuals. A) The frequency of CD8⁺ T cells co-expressing PD-1 and 2B4 was significantly higher in ART-naïve individuals compared with individuals receiving ART, independent of when ART was initiated. B) The frequency of CD8⁺ T cells co-expressing PD-1, 2B4 and CD160 was significantly higher in ART-naïve individuals compared with ART-treated individuals and HIV-negative individuals, independent of the time of treatment initiation. ** means 0.001</p

The CAF05 adjuvant enhances the cytotoxicity of CD8 T cells.

<p>Mice were immunized three times i.p. with 20 µg Gag p24 protein alone (‘no adjuvant’) or adjuvanted in either CAF01 or CAF05. Three weeks later <i>in vivo</i> cytotoxicity was determined 1 day after adoptive transfer of AMQMLKETI-pulsed (CFSE^hi) or unpulsed (CFSE^lo) CFSE-stained target cells from naïve mice. (A) The specific lysis relative to the naïve control group. Bars represent the mean specific lysis of individual mice within each group (n = 3/4+/− SEM). Significant differences as determined by one-way ANOVA followed by Tukey’s post test have been indicated: *<i>p<</i>0.05, ***<i>p<</i>0.001. (B) Gating strategy for one representative mouse. (C) Representative histograms of CFSE^lo and CFSE^hi populations for all three immunization groups.</p

Peptide pool matrix.

1<p>Gag p24 15-mer peptides ARP7111.34 to ARP7111.88 were numbered from 1–55 and assigned to 15 pools according to the matrix.</p>2<p><i>NA</i>, not applicable.</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

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

2B4 and PD-1 in individuals on ART normalizes to the levels observed for HIV-negative controls.

<p>Frequencies of total and memory CD8⁺ T cells expressing the inhibitory receptors 2B4, PD-1 and CD160. A) The frequency of CD8⁺ T cells expressing 2B4 was significantly higher in ART-naïve individuals compared with ART-treated individuals and the HIV-negative controls, independent of when ART was initiated. B) The frequency of CD8⁺ T cells expressing PD-1 was significantly higher in ART-naïve individuals compared with ART-treated individuals and the HIV-negative controls, independent of when ART was initiated. C) No differences were found in the frequency of CD8⁺ T cells expressing CD160 between ART-naïve, ART-treated and HIV-negative individuals. * means 0.01</p