Immunological Memory Transferred with CD4 T Cells Specific for Tuberculosis Antigens Ag85B-TB10.4: Persisting Antigen Enhances Protection

BACKGROUND:High levels of death and morbidity worldwide caused by tuberculosis has stimulated efforts to develop a new vaccine to replace BCG. A number of Mycobacterium tuberculosis (Mtb)-specific antigens have been synthesised as recombinant subunit vaccines for clinical evaluation. Recently a fusion protein of TB antigen Ag85B combined with a second immunodominant TB antigen TB10.4 was emulsified with a novel non-phospholipid-based liposomal adjuvant to produce a new subunit vaccine, investigated here. Currently, there is no consensus as to whether or not long-term T cell memory depends on a source of persisting antigen. To explore this and questions regarding lifespan, phenotype and cytokine patterns of CD4 memory T cells, we developed an animal model in which vaccine-induced CD4 memory T cells could transfer immunity to irradiated recipients. METHODOLOGY/PRINCIPAL FINDINGS:The transfer of protective immunity using Ag85B-TB10.4-specific, CD45RB(low) CD62L(low) CD4 T cells was assessed in sub-lethally irradiated recipients following challenge with live BCG, used here as a surrogate for virulent Mtb. Donor T cells also carried an allotype marker allowing us to monitor numbers of antigen-specific, cytokine-producing CD4 T cells in recipients. The results showed that both Ag85B-TB10.4 and BCG vaccination induced immunity that could be transferred with a single injection of 3x10(6) CD4 T cells. Ten times fewer numbers of CD4 T cells (0.3x10(6)) from donors immunised with Ag85B-TB10.4 vaccine alone, transferred equivalent protection. CD4 T cells from donors primed by BCG and boosted with the vaccine similarly transferred protective immunity. When BCG challenge was delayed for 1 or 2 months after transfer (a test of memory T cell survival) recipients remained protected. Importantly, recipients that contained persisting antigen, either live BCG or inert vaccine, showed significantly higher levels of protection (p<0.01). Overall the numbers of IFN-gamma-producing CD4 T cells were poorly correlated with levels of protection. CONCLUSIONS/SIGNIFICANCE:The Ag85B-TB10.4 vaccine, with or without BCG-priming, generated TB-specific CD4 T cells that transferred protective immunity in mice challenged with BCG. The level of protection was enhanced in recipients containing a residual source of specific antigen that could be either viable or inert

Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid a-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-?) and interleukin-4 (IL-4). However, aGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the aGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-? secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs

Kinetics of the antigen-specific CD4 T cell response in BCG-immunised mice after a boost with Ag85B-TB10.4.

<p>Mice were killed at selected days following antigen injection and spleen cells analysed for ICC IFN-γ. Results are the net percentage of IFN-γ⁺ CD4 T cells stimulated in the presence of Ag85B-TB10.4 after subtraction of the response without antigen. The results were pooled from 8 experiments. Each point represents the mean+SD of 3 or 4 mice, except day 3, n = 2.</p

Protective immunity persists for at least 8 weeks following transfer of prime/boost CD4 T cells.

<p>(A) CD45RB^low CD4 T cells (3×10⁶) from donors primed with BCG and boosted with Ag85B-TB10.4 (Pr/Boost) or 3×10⁶ CD4 T cells from unprimed donors (Naïve) or no cells (None) were transferred to irradiated recipients and challenged with BCG immediately (d 1) or 8 weeks later (d 56) (B, C). Three weeks after challenge spleen cells were assayed for BCG CFUs (B) and ICC IFN-γ⁺ donor CD4 T cells (C). The results are from one experiment. Each point represents a single recipient. Horizontal bars or histograms + SD are means of 4 recipients/group. ** p<0.01.</p

Persisting antigen enhances protection.

<p>(A) To establish a potential source of residual antigen (Ag) mice were injected with BCG or Ag85B-TB10.4, 4 weeks (d -28) before irradiation and cell transfer. Control recipients received no source of residual Ag (None) and were irradiated 1 day before cell transfer. CD45RB^low CD4 T cells (3×10⁶) from BCG primed/Ag85B-TB10.4 boosted donors (Pr/Boost, solid symbols) or unprimed CD4 T cells (Naïve, open symbols) were transferred into recipients after irradiation on d 0. All recipients were challenged with BCG 4 weeks later (d 28). (B) Three weeks after challenge spleen cells were assayed for BCG CFUs. Each point represents a single recipient. Horizontal bars are means of 4 recipients/group. ** p<0.01.</p

Differential saliva-induced breakdown of starch filled protein gels in relation to sensory perception

In this study, the differential breakdown of protein gels containing four types of high and low cross-linked starch granules were studied. Susceptibility to saliva-induced breakdown of starch granules and the consequences of these for overall breakdown of the gel matrix were captured using a multiple extrusion cell (MEC). Gels filled with two types of starch were used for sensorial evaluation by a QDA panel and the mechanical impact of the starch granules on these latter gels was characterized using uniaxial compression measurements. These data were used to better understand differences in sensory mouth feel attributes. MEC measurements indicated that the low cross-linked starches were more susceptible to saliva-induced breakdown compared to their highly cross-linked counterparts. The sensory space of starch filled gels was divided into three dimensions. Protein content of the gel matrix determined one dimension, resulting in high sensory ratings for separating and slippery mouth feel. The distinction into the two other dimensions, one being dominated by grainy/spreadable/sticky and the other by crumbly/crumbly effort, originates mainly from different starch types used: larger starch granules from the low cross-linked potato starch were perceived as more grainy and gels filled with these granules were more spreadable with a lower rating for crumbly and crumbly effort. Surprisingly, in most cases ratings for firm for both starch types were more or less comparable, indicating that these granules behave as inert fillers in a rather similar way, as also suggested by compression measurements. This work indicates that low cross-linked potato starch displays a higher susceptibility for digestion by amylase present in saliva. This is possibly the reason for the larger spreadability and lower rating for crumbly effort of the gel containing this type of starch