Immunocorrelates of CAF family adjuvants.

The development of the CAF family adjuvant was initiated around 20 years ago when Statens Serum Institut was preparing its first generation protein based recombinant subunit vaccine against tuberculosis for clinical testing, but realized that there were no clinically relevant adjuvants available that would support the strong CMI response needed. Since then the aim for the adjuvant research at Statens Serum Institut has been to provide adjuvants with distinct immunogenicity profiles correlating with protection for any given infectious disease. Two of the adjuvants CAF01 and CAF09 are currently being evaluated in human clinical trials. The purpose of this review is to give an overview of the immunocorrelates of those CAF adjuvants furthest in development. We further aim at giving an overview of the mechanism of action of the CAF adjuvants.

Adjuvants Based on Synthetic Mycobacterial Cord Factor Analogues: Biophysical Properties of Neat Glycolipids and Nanoself-Assemblies with DDA.

Synthetic mycobacterial cord factor analogues, e.g., trehalose 6,6'-dibehenate (TDB), are highly promising adjuvants due to their strong immunopotentiating capabilities, but their biophysical properties have remained poorly characterized. Here, we report the synthesis of an array of synthetic TDB analogues varying in acyl chain length, degree of acylation, and headgroup display, which was subjected to biophysical characterization of neat nondispersed self-assembled nanostructures in excess buffer and as aqueous dispersions with cationic dimethyldioctadecylammonium (DDA) bromide. The array comprised trehalose mono- (TMX) and diester (TDX) analogues with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), myristate (Myr), and laurate (L)] and an analogue with a short hydrophilic polyethylene glycol (PEG) linker inserted between the trehalose headgroup of TDS and the acyl chains (PEG-TDS). All dispersions were liposomes, but in contrast to the colloidally stable and highly cationic TDX-containing liposomes, the zeta-potential was significantly reduced for DDA/TMX and DDA/PEG-TDS liposomes, suggesting a charge-shielding effect, which compromises the colloidal stability. An increased d-spacing was observed for the lamellar phase of neat TDB analogues in excess buffer (TDS < TMS < PEG-TDS), confirming that the charge shielding is caused by an extended molecular configuration of the more flexible headgroup. Differential scanning calorimetry showed highly cooperative phase transitions for all tested dispersions albeit the monoesters destabilized the lipid bilayers. Langmuir experiments demonstrated that incorporation of TDXs and PEG-TDS stabilized DDA monolayers due to improved hydrogen bonding and reduced intermolecular repulsions. In conclusion, data suggest that the DDA/TDS dispersions exhibit favorable physicochemical properties rendering these DDA/TDS liposomes an attractive vaccine adjuvant, and they emphasize that not only the receptor binding and immune activation but also the biophysical properties of immunopotentiator formulations should be collectively considered when designing adjuvants with optimal safety, efficacy, and storage stability.

Liposomal T cell engager and re-director for tumor cell eradication in cancer immunotherapy.

T cells are one of the most important effector cells in cancer immunotherapy. Various T cell-dependent bispecific antibody (TDB) drugs that engage T cells for targeted cancer cell lysis are being developed. Here, we describe supra-molecular T-cell redirecting antibody fragment-anchored liposomes (TRAFsomes) and report their immune modulation and anti-cancer effects. We found that TRAFsomes containing different copies of anti-CD3 fragments displayed different T cell modulation profiles, showing that optimization of surface density is needed to define the therapeutic window for potentiating cancer cell-specific immune reactions while minimizing nonspecific side effects. Moreover, small molecular immunomodulators may also be incorporated by liposomal encapsulation to drive CD8 + T cell biased immune responses. In vivo studies using human peripheral blood mononuclear cell reconstituted mouse models showed that TRAFsomes remained bounded to human T cells and persisted for more than 48 hours after injection. However, only TRAFsomes containing a few anti-CD3 (n = 9) demonstrated significant T cell-mediated anti-cancer activities to reverse tumor growth. Those with more anti-CD3s (n = 70) caused tumor growth and depletion of human T cells at the end of treatments. These data suggested that TRAFsomes can be as potent as traditional TDBs and the liposomal structure offers great potential for immunomodulation and improvement of the therapeutic index.Abbreviation: Chimeric antigen receptor T cells (CAR-T cells), Cytokine release syndrome (CRS) Cytotoxic T cell (CTL) Effector: target ratios (E:T ratios), Heavy chain (HC) Immune-related adverse events (irAE), Large unilamellar vesicle (LUV), Peripheral blood mononuclear cells (PBMCs, Single-chain variable fragment (scFv), T cell-dependent bispecific antibody (TDB), T cell redirecting antibody fragment-anchored liposomes (TRAFsomes), Methoxy poly-(ethylene glycol) (mPEG).

Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine.

Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8(+) epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6'-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4(+) Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ(+) CD8(+) T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8(+) T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.