Seasonal influenza split vaccines confer partial cross-protection against heterologous influenza virus in ferrets when combined with the CAF01 adjuvant

Influenza epidemics occur annually, and estimated 5–10% of the adult population and 20–30% of children will become ill from influenza infection. Seasonal vaccines primarily work through the induction of neutralizing antibodies against the principal surface antigen hemagglutinin (HA). This important role of HA-specific antibodies explains why previous pandemics have emerged when new HAs have appeared in circulating human viruses. It has long been recognized that influenza virus-specific CD4(+) T cells are important in protection from infection through direct effector mechanisms or by providing help to B cells and CD8(+) T cells. However, the seasonal influenza vaccine is poor at inducing CD4(+) T-cell responses and needs to be combined with an adjuvant facilitating this response. In this study, we applied the ferret model to investigate the cross-protective efficacy of a heterologous trivalent influenza split-virion (TIV) vaccine adjuvanted with the CAF01 adjuvant, with proven ability to induce CD4(+) T-cell and antibody responses in mice, ferrets, pigs, primates, and humans. Our results indicate that CAF01-adjuvanted vaccine induces HA inhibition (HAI)-independent protection after heterologous challenge, manifested as reduced viral load and fever. On the other hand, we observe increased inflammation in the airways and more neutrophil and mononuclear cell infiltration in these ferrets when compared with optimally protected animals, i.e., ferrets receiving the same vaccine but a homologous challenge. This suggest that HAI-independent immunity induced by TIV + CAF01 can reduce viral shedding and systemic disease symptoms, but does not reduce local inflammation in the nasal cavity

Cationic Liposomes Containing Mycobacterial Lipids: a New Powerful Th1 Adjuvant System

The immunostimulation provided by the mycobacterial cell wall has been exploited for many decades, e.g., in Freund's complete adjuvant. Recently, the underlying mechanism behind this adjuvant activity, including Toll receptor signaling, has begun to be unraveled, confirming the potential of mycobacterial constituents to act as adjuvants. In this study, the immunostimulatory properties of a Mycobacterium bovis BCG lipid extract were tested for their adjuvant activity. Administration of the lipids in dimethyl dioctadecyl ammonium bromide-based cationic liposomes induced a powerful Th1 response characterized by markedly elevated antigen-specific immunoglobulin G2a (IgG2a) isotype antibodies and substantial production of gamma interferon. The adjuvant formulation (designated mycosomes) elicited high levels of gamma interferon both in C57BL/6 as well as in Th2-prone BALB/c mice. Furthermore, the mycosomes induced immune responses to protein antigens from several sources including Mycobacterium tuberculosis, Chlamydia muridarum, and tetanus toxoid. In a tuberculosis challenge model, the mycosomes combined with the Ag85B-ESAT-6 fusion protein were demonstrated to have a unique ability to maintain sustained immunological memory at a level superior to live BCG

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

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

Stimulation of PBMCs with individual peptides selected based on positive peptide pools.

<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 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. ND, not done. NA, not applicable (peptide 44 could not be dissolved).</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