Kevin G. J. Pollock

Analysis of the role of vaccine adjuvants in modulating dendritic cell activation and antigen presentation in vitro

We have studied the effects of adjuvant formulations on the activation and antigen-presenting functions of bone marrow-derived dendritic cells (DCs). While LPS could induce high-level expression of MHC Class II and co-stimulator molecules on DCs, it did not enhance antigen presentation to co-stimulation independent DO11.GFP T hybridoma cells. In contrast, alum, NISV and PLGA formulations failed to activate DCs, but NISV and PLGA could enhance antigen-presentation efficiency by 10-100-fold. Irrespective of the previously described antigen release characteristics of each adjuvant, antigen presentation peaked at 6h and waned thereafter for all formulations. Given the importance of DCs in the activation of nai;ve T cell responses, these studies suggest that as yet undefined pathways of DC activation in vivo may underlie the activity of alum, PLGA and NISV adjuvants. Furthermore, as NISV and PLGA do not appear to act as slow-release systems in DCs, the ability of these particulate systems to induce high levels of antigen presentation by DCs probably has a more significant role in their adjuvant activity.

Interleukin‐18 plays a role in both the alum‐induced T helper 2 response and the T helper 1 response induced by alum‐adsorbed interleukin‐12

Previous studies have shown that the antigen‐specific T helper 2 (Th2) response induced by alum adjuvants is interleukin (IL)‐4 independent. As a role for IL‐18 in Th2 induction has recently been described, in addition to its role in enhancing Th1 responses, we have studied the Th2 response induced by ovalbumin (OVA) adsorbed to alum in wild‐type and IL‐18‐deficient mice. Our results indicate that while endogenous IL‐18 facilitates alum‐induced IL‐4 production, OVA‐specific immunoglobulin G1 (IgG1) and IgE production remain unaffected. Furthermore, antigen‐specific Th1 responses induced with alum/IL‐12‐adsorbed OVA were demonstrated to be highly IL‐18 dependent. Despite these observations, injection of BALB/c mice with exogenous IL‐18 adsorbed to alum/OVA did not alter IL‐4 or interferon‐γ production by T cells and had little effect on the relative production of IgG1/IgG2a antibody subclasses compared with alum/OVA inoculated mice. However, the previously described synergism between IL‐12 and IL‐18 in Th1 induction was evident as the Th1‐promoting activity of alum/IL‐12 against adsorbed OVA was greatly augmented by the coadministration of IL‐18. These results indicate that while alum‐induced IL‐18 can facilitate Th2 induction, the addition of exogenous IL‐18 cannot further enhance the alum‐induced Th2 response.

Vesicle Size Influences the Trafficking, Processing, and Presentation of Antigens in Lipid Vesicles

Although it is accepted that particulate Ags are more immunogenic than soluble Ags in vivo, it is unclear whether this effect can be explained solely through enhanced uptake by APCs. In this study we demonstrate that vesicle size modulated the efficiency of Ag presentation by murine macrophages and that this effect was accompanied by a profound change in trafficking of Ag. Ag prepared in large particles (560 nm) was delivered into early endosome-like, immature phagosomes, whereas smaller vesicles (155 nm) and soluble Ags localized rapidly to late endosomes/lysosomes. However, peptide/class II complexes could be detected in both compartments. Phagosomes formed on uptake of large vesicles recruit Ag-processing apparatus while retaining the characteristics of early endosomes. In contrast, smaller vesicles bypassed this compartment, appeared to go more rapidly to lysosomal compartments, and exhibited reduced Ag-presenting efficiency. We conclude that the ability of phagocytosed, particulate Ag to target early phagosomes results in more efficient Ag presentation.

The Leishmania mexicana cysteine protease, CPB2.8, induces potent Th2 responses

We have previously identified that Leishmania mexicana cysteine proteases (CPs) are virulence factors. We have now produced a recombinant L. mexicana CP, CPB2.8, which has similar enzymatic activity to native enzyme. Inoculation of CPB2.8 (≤5 μg) into the footpads of BALB/c mice not only up-regulated mRNA transcripts for IL-4 and IL-4 production in the draining popliteal lymph nodes, but also polarized splenocyte anti-CD3 stimulated responses toward a Th2 bias as measured by increased IL-5 production compared with controls. In agreement with promoting a Th2 response, CPB2.8 also induced strong specific IgE responses in treated mice as well as increasing whole IgE levels. Inhibition of the enzyme activity of CPB2.8 by treatment with E-64 ablated the enzyme’s ability to induce IgE. Significantly, infection of mice with CPB-deficient parasites failed to stimulate production of IgE, unlike infection with wild-type parasites. Furthermore, enzymatically active (<0.1 U/ml) but not E-64-inactivated CPB2.8 was able to proteolytically cleave CD23 and CD25, although not B220 or CD4 from murine lymphocytes. These properties are similar to those demonstrated by the house dust mite allergen Der p I and provide an explanation for the immunomodulatory activity of the CPB2.8 virulence factor. Vaccination with CPB2.8 enhanced L. mexicana lesion growth compared with control animals. Nevertheless, vaccination with IL-12 and CPB2.8 resulted in a degree of protection associated with inhibition of lesion growth and a Th1 response. Thus, CPB2.8 is a potent Th2-inducing molecule capable of significant vaccine potential if administered with a suitable adjuvant.

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.

Crystallization and preliminary X-ray analysis of shikimate dehydrogenase from Escherichia coli

Shikimate dehydrogenase from Escherichia coli has been crystallized by the vapour-diffusion method using ammonium sulfate as a precipitant. Mass spectrometry confirmed the purity of the enzyme and dynamic light scattering was used to find the appropriate additives to yield a monodisperse enzyme solution. The crystals are monoclinic, space group C2, with unit-cell parameters a = 110.0, b = 139.8, c = 102.6 A, beta = 122.2 degrees (at 100 K). Native crystals diffract to 2.3 A in-house on a rotating-anode X-ray source. The asymmetric unit is likely to contain four molecules, related by 222 symmetry, corresponding to a packing density of 2.86 A(3) Da(-1).