Protection against experimental cryptococcosis elicited by Cationic Adjuvant Formulation 01-adjuvanted subunit vaccines.

The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4+ T cells which produced interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8+ T cells were also found, albeit in lesser numbers. A significant, durable IFNγ response was observed in the lungs, spleen, and blood. Moreover, IFNγ secretion following ex vivo stimulation directly correlated with fungal control in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma.

BACKGROUND: Neoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX-01-specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose-escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX-01 in addition to anti-PD-1 therapy. METHODS: Patients with metastatic melanoma on anti-PD-1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor-derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX-01. Vaccines were administered at 2-week intervals for a total of three intraperitoneal and three intramuscular injections. The study's primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates. RESULTS: Compared with the base dose level previously reported, no new vaccine-related serious adverse events were observed during dose escalation of EVX-01 in combination with an anti-PD-1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX-01 induced peptide-specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX-01 peptides and the PIONEER quality score. CONCLUSION: Immunization with EVX-01-CAF09b in addition to anti-PD-1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen-specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX-01 in combination with anti-PD-1 therapy.

Protection against experimental cryptococcosis elicited by Cationic Adjuvant Formulation 01-adjuvanted subunit vaccines.

The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4+ T cells which produced Interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8+ T cells were also found, albeit in lesser numbers. A significant, durable IFNγ response was observed in the lungs, spleen, and blood. Moreover, IFNγ secretion following ex vivo stimulation directly correlated with fungal clearance in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials.

Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAF®01 or CDA.

Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of causing a systemic infection called Glässer's disease. This disease is more frequent in young post-weaning piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inactivated vaccines, which promote limited cross-protection against different serovars. For this reason, there is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic adjuvant CAF®01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non-immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAF®01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF®01 controlled more efficiently a natural nasal colonization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental procedure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer's disease vaccine and could contribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization.

Method of manufacturing CAF®09 liposomes affects immune responses induced by adjuvanted subunit proteins.

The ability to induce antigen-specific CD4+ and CD8+T-cell responses is one of the fundamental requirements when developing new efficacious vaccines against challenging infectious diseases and cancer. However, no adjuvants are currently approved for human subunit vaccines that induce T-cell immunity. Here, we incorporated a Toll-like receptor 4 agonist, i.e., the ionizable lipidoid L5N12, in the liposomal cationic adjuvant formulation 09 (CAF®09), and found that modified CAF®09 liposomes possess preserved adjuvant function as compared to unmodified CAF®09. CAF®09 consists of the cationic lipid dimethyldioctadecylammonium (DDA), monomycoloyl glycerol analogue 1 (MMG-1), and polyinosinic:polycytidylic acid [poly(I:C)]. By using the microfluidic mixing technology for liposome preparation, we gradually replaced DDA with L5N12, while keeping the molar ratios of MMG-1 and poly(I:C) constant. We found that this type of modification resulted in colloidally stable liposomes, which were significantly smaller and displayed reduced surface charge as compared to unmodified CAF®09, prepared by using the conventional thin film method. We showed that incorporation of L5N12 decreases the membrane rigidity of CAF®09 liposomes. Furthermore, vaccination with antigen adjuvanted with L5N12-modified CAF®09 or antigen adjuvanted with unmodified CAF®09, respectively, induced comparable antigen-specific serum antibody titers. We found that antigen adjuvanted with L5N12-modified CAF®09 induced antigen-specific effector and memory CD4+ and CD8+T-cell responses in the spleen comparable to those induced when unmodified CAF®09 was used as adjuvant. However, incorporating L5N12 did not have a synergistic immunopotentiating effect on the antibody and T-cell responses induced by CAF®09. Moreover, vaccination with antigen adjuvanted with unmodified CAF®09, which was manufactured by using microfluidic mixing, induced significantly lower antigen-specific CD4+ and CD8+T-cell responses than vaccination with antigen adjuvanted with unmodified CAF®09, which was prepared by using the thin film method. These results show that the method of manufacturing affects CAF®09 liposome adjuvanted antigen-specific immune responses, which should be taken into consideration when evaluating immunogenicity of subunit protein vaccines.

First in man study: Bcl-Xl_42-CAF®09b vaccines in patients with locally advanced prostate cancer.

Background: The B-cell lymphoma-extra-large (Bcl-XL) protein plays an important role in cancer cells' resistance to apoptosis. Pre-clinical studies have shown that vaccination with Bcl-XL-derived peptides can induce tumor-specific T cell responses that may lead to the elimination of cancer cells. Furthermore, pre-clinical studies of the novel adjuvant CAF®09b have shown that intraperitoneal (IP) injections of this adjuvant can improve the activation of the immune system. In this study, patients with hormone-sensitive prostate cancer (PC) received a vaccine consisting of Bcl-XL-peptide with CAF®09b as an adjuvant. The primary aim was to evaluate the tolerability and safety of IP and intramuscular (IM) administration, determine the optimal route of administration, and characterize vaccine immunogenicity. Patients and methods: Twenty patients were included. A total of six vaccinations were scheduled: in Group A (IM to IP injections), ten patients received three vaccines IM biweekly; after a three-week pause, patients then received three vaccines IP biweekly. In Group B (IP to IM injections), ten patients received IP vaccines first, followed by IM under a similar vaccination schedule. Safety was assessed by logging and evaluating adverse events (AE) according to Common Terminology Criteria for Adverse Events (CTCAE v. 4.0). Vaccines-induced immune responses were analyzed by Enzyme-Linked Immunospot and flow cytometry. Results: No serious AEs were reported. Although an increase in T cell response against the Bcl-XL-peptide was found in all patients, a larger proportion of patients in group B demonstrated earlier and stronger immune responses to the vaccine compared to patients in group A. Further, we demonstrated vaccine-induced immunity towards patient-specific CD4, and CD8 T cell epitopes embedded in Bcl-XL-peptide and an increase in CD4 and CD8 T cell activation markers CD107a and CD137 following vaccination. At a median follow-up of 21 months, no patients had experienced clinically significant disease progression. Conclusion: The Bcl-XL-peptide-CAF®09b vaccination was feasible and safe in patients with l hormone-sensitive PC. In addition, the vaccine was immunogenic and able to elicit CD4 and CD8 T cell responses with initial IP administration eliciting early and high levels of vaccine-specific responses in a higher number og patients. Clinical trial registration: https://clinicaltrials.gov, identifier NCT03412786.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity.

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

CAF08 adjuvant enables single dose protection against respiratory syncytial virus infection in murine newborns.

Respiratory syncytial virus is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of Th 1 immunity. Here we show application of cationic adjuvant formulation CAF08, a liposomal vaccine formulation tailored to induce Th 1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. We apply quantitative phosphoproteomics to human dendritic cells and reveal a role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identify signaling events resulting in antigen cross-presentation. In a murine in vivo model a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protects newborn mice from RSV infection by induction of antigen-specific CD8+ T-cells and Th1 cells. Overall, we describe a pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other respiratory viral pathogens.

A Mutated Prostatic Acid Phosphatase (PAP) Peptide-Based Vaccine Induces PAP-Specific CD8+ T Cells with Ex Vivo Cytotoxic Capacities in HHDII/DR1 Transgenic Mice.

BACKGROUND: Current treatments for castrate (hormone)-resistant prostate cancer (CRPC) remain limited and are not curative, with a median survival from diagnosis of 23 months. The PAP-specific Sipuleucel-T vaccine, which was approved by the FDA in 2010, increases the Overall Survival (OS) by 4 months, but is extremely expensive. We have previously shown that a 15 amino accid (AA) PAP sequence-derived peptide could induce strong immune responses and delay the growth of murine TRAMP-C1 prostate tumors. We have now substituted one amino acid and elongated the sequence to include epitopes predicted to bind to several additional HLA haplotypes. Herein, we present the immunological properties of this 42mer-mutated PAP-derived sequence (MutPAP42mer). METHODS: The presence of PAP-135-143 epitope-specific CD8+ T cells in the blood of patients with prostate cancer (PCa) was assessed by flow cytometry using Dextramer™ technology. HHDII/DR1 transgenic mice were immunized with mutated and non-mutated PAP-derived 42mer peptides in the presence of CAF®09 or CpG ODN1826 (TLR-9 agonist) adjuvants. Vaccine-induced immune responses were measured by assessing the proportion and functionality of splenic PAP-specific T cells in vitro. RESULTS: PAP-135-143 epitope-specific CD8+ T cells were detected in the blood of patients with PCa and stimulation of PBMCs from patients with PCa with mutPAP42mer enhanced their capacity to kill human LNCaP PCa target cells expressing PAP. The MutPAP42mer peptide was significantly more immunogenic in HHDII/DR1 mice than the wild type sequence, and immunogenicity was further enhanced when combined with the CAF®09 adjuvant. The vaccine induced secretory (IFNγ and TNFα) and cytotoxic CD8+ T cells and effector memory splenic T cells. CONCLUSIONS: The periphery of patients with PCa exhibits immune responsiveness to the MutPAP42mer peptide and immunization of mice induces/expands T cell-driven, wild-type PAP immunity, and therefore, has the potential to drive protective anti-tumor immunity in patients with PCa.

Personalized therapy with peptide-based neoantigen vaccine (EVX-01) including a novel adjuvant, CAF®09b, in patients with metastatic melanoma.

The majority of neoantigens arise from unique mutations that are not shared between individual patients, making neoantigen-directed immunotherapy a fully personalized treatment approach. Novel technical advances in next-generation sequencing of tumor samples and artificial intelligence (AI) allow fast and systematic prediction of tumor neoantigens. This study investigates feasibility, safety, immunity, and anti-tumor potential of the personalized peptide-based neoantigen vaccine, EVX-01, including the novel CD8+ T-cell inducing adjuvant, CAF®09b, in patients with metastatic melanoma (NTC03715985). The AI platform PIONEERTM was used for identification of tumor-derived neoantigens to be included in a peptide-based personalized therapeutic cancer vaccine. EVX-01 immunotherapy consisted of 6 administrations with 5-10 PIONEERTM-predicted neoantigens as synthetic peptides combined with the novel liposome-based Cationic Adjuvant Formulation 09b (CAF®09b) to strengthen T-cell responses. EVX-01 was combined with immune checkpoint inhibitors to augment the activity of EVX-01-induced immune responses. The primary endpoint was safety, exploratory endpoints included feasibility, immunologic and objective responses. This interim analysis reports the results from the first dose-level cohort of five patients. We documented a short vaccine manufacturing time of 48-55 days which enabled the initiation of EVX-01 treatment within 60 days from baseline biopsy. No severe adverse events were observed. EVX-01 elicited long-lasting EVX-01-specific T-cell responses in all patients. Competitive manufacturing time was demonstrated. EVX-01 was shown to be safe and able to elicit immune responses targeting tumor neoantigens with encouraging early indications of a clinical and meaningful antitumor efficacy, warranting further study.

Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines.

One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.

Applying Microfluidics for the Production of the Cationic Liposome-Based Vaccine Adjuvant CAF09b.

Subunit vaccines require particulate adjuvants to induce the desired immune responses. Pre-clinical manufacturing methods of adjuvants are often batch dependent, which complicates scale-up for large-scale good manufacturing practice (GMP) production. The cationic liposomal adjuvant CAF09b, composed of dioctadecyldimethylammonium bromide (DDA), monomycoloyl glycerol analogue 1 (MMG) and polyinosinic:polycytidylic acid [poly(I:C)], is currently being clinically evaluated in therapeutic cancer vaccines. Microfluidics is a promising new method for large-scale manufacturing of particle-based medicals, which is scalable from laboratory to GMP production, and a protocol for production of CAF09b by this method was therefore validated. The influence of the manufacture parameters [Ethanol] (20-40% v/v), [Lipid] (DDA and MMG, 6-12 mg/mL) and dimethyl sulfoxide [DMSO] (0-10% v/v) on the resulting particle size, colloidal stability and adsorption of poly(I:C) was evaluated in a design-of-experiments study. [Ethanol] and [DMSO] affected the resulting particle sizes, while [Lipid] and [DMSO] affected the colloidal stability. In all samples, poly(I:C) was encapsulated within the liposomes. At [Ethanol] 30% v/v, most formulations were stable at 21 days of manufacture with particle sizes <100 nm. An in vivo comparison in mice of the immunogenicity to the cervical cancer peptide antigen HPV-16 E7 adjuvanted with CAF09b prepared by lipid film rehydration or microfluidics showed no difference between the formulations, indicating adjuvant activity is intact. Thus, it is possible to prepare suitable formulations of CAF09b by microfluidics.

Modulation of immune responses using adjuvants to facilitate therapeutic vaccination.

Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies.

Cutting Edge: TNF Is Essential for Mycobacteria-Induced MINCLE Expression, Macrophage Activation, and Th17 Adjuvanticity.

TNF blockade is a successful treatment for human autoimmune disorders like rheumatoid arthritis and inflammatory bowel disease yet increases susceptibility to tuberculosis and other infections. The C-type lectin receptors (CLR) MINCLE, MCL, and DECTIN-2 are expressed on myeloid cells and sense mycobacterial cell wall glycolipids. In this study, we show that TNF is sufficient to upregulate MINCLE, MCL, and DECTIN-2 in macrophages. TNF signaling through TNFR1 p55 was required for upregulation of these CLR and for cytokine secretion in macrophages stimulated with the MINCLE ligand trehalose-6,6-dibehenate or infected with Mycobacterium bovis bacillus Calmette-Guérin. The Th17 response to immunization with the MINCLE-dependent adjuvant trehalose-6,6-dibehenate was specifically abrogated in TNF-deficient mice and strongly attenuated by TNF blockade with etanercept. Together, interference with production or signaling of TNF antagonized the expression of DECTIN-2 family CLR, thwarting vaccine responses and possibly increasing infection risk.

Injection Vaccines Formulated with Nucleotide, Liposomal or Mineral Oil Adjuvants Induce Distinct Differences in Immunogenicity in Rainbow Trout.

Protection facilitated by the widespread use of mineral oil adjuvanted injection vaccines in salmonid fish comes with adverse effects of varying severity. In this study, we characterized the immunological profiles of two alternative vaccine formulations, both with proven efficacy and an improved safety profile in rainbow trout. Experimental injection vaccines were prepared on an identical whole-cell Aeromonas salmonicida bacterin platform and were formulated with CpG oligodeoxynucleotides, a liposomal (CAF01) or a benchmark mineral oil adjuvant, respectively. A naïve group, as well as bacterin and saline-injected groups were also included. Following administration, antigen-specific serum antibody titers, the tissue distribution of immune cell markers, and the expression of immune-relevant genes following the in vitro antigenic restimulation of anterior kidney leukocytes was investigated. Immunohistochemical staining suggested prolonged antigen presentation for the particulate formulations and increased mucosal presence of antigen-presenting cells in all immunized fish. Unlike the other immunized groups, the CAF01 group only displayed a transient elevation in specific antibody titers and immunohistochemical observations, and the transcription data suggest an increased role of cell-mediated immunity for this group. Finally, the transcription profile of the CpG formulation approached that of a TH1 profile. When compared to the benchmark formulation, CAF01 and CpG adjuvants induce slight, but distinct differences in the resulting protective immune responses. This is important, as it allows a broader immunological approach for the future development of safer vaccines.

Toll like-receptor agonist Pam3Cys modulates the immunogenicity of liposomes containing the tuberculosis vaccine candidate H56.

A major roadblock in the development of novel vaccines is the formulation and delivery of the antigen. Liposomes composed of a dimethyldioctadecylammonium (DDA) backbone and the adjuvant trehalose-6-6-dibehenate (TDB, termed "cationic adjuvant formulation (CAF01)", promote immunogenicity and protective efficacy of vaccines, most notably against infection with Mycobacterium tuberculosis. Specifically, the multicomponent antigen H56 delivered by CAF01 protects against tuberculosis in mice. Here we investigated whether the inclusion of immune-modulatory adjuvants into CAF01 modulates the immunogenicity of H56/CAF01 in vitro and in vivo. Based on our recent findings we selected the active sequence of the mycobacterial 19 kDa lipoprotein, Pam3Cys, which interacts with Toll like receptor 2 to induce an antimicrobial pathway. H56/CAF01-Pam3Cys liposomes were characterized for Pam3Cys incorporation, size, toxicity and activation of primary human macrophages. Macrophages efficiently take up H56/CAF01-Pam3Cys and trigger the release of significantly higher levels of TNF, IL-12 and IL-10 than H56/CAF01 alone. To evaluate the immunogenicity in vivo, we immunized mice with H56/CAF01-Pam3Cys and measured the release of IFN-γ and IL-17A by lymph node cells and spleen cells. While the antigen-specific production of IFN-γ was reduced by inclusion of Pam3Cys into H56/CAF01, the levels of IL-17A remained unchanged. In agreement with this finding, the concentration of the IFN-γ-associated IgG2a antibodies in the serum was lower than in H56/CAF01 immunized animals. These results provide proof of concept that Toll like-receptor agonist can be included into liposomes to modulate immune responses. The discordant results between the in vitro studies with human macrophages and in vivo studies in mice highlight the relevance and complexity of comparing immune responses in different species.

Comparison of two different PEGylation strategies for the liposomal adjuvant CAF09: Towards induction of CTL responses upon subcutaneous vaccine administration.

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG1000 moieties, resulting in mono- and bis-PEG-peptides of different lengths (10, 15 and 20 amino acid residues). For comparison, CAF09 was also grafted by inclusion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG2000) in the bilayer structure during preparation. Grafting of CAF09 with either type of PEG resulted in charge shielding, evident from a reduced surface charge. Upon s.c. immunization of mice with the model antigen ovalbumin (OVA) adjuvanted with PEGylated CAF09, stronger CTL responses were induced as compared to immunization of mice with unadjuvanted OVA. Biodistribution studies confirmed that grafting of CAF09 with DSPE-PEG2000 improved the passive drainage of the vaccine to LNs, because a higher dose fraction was recovered in DCs present in the draining LNs, as compared to the dose fraction detected for non-PEGylated CAF09. In conclusion, PEGylation of CAF09 may be a useful strategy for the design of an adjuvant, which induces CTL responses after s.c. and i.m. administration. In the present studies, CAF09 grafted with 10 mol% DSPE-PEG2000 is the most promising of the tested adjuvants, but additional studies are required to further elucidate the potential of the strategy.

Tuning the Two-Dimensional Electron Gas at Oxide Interfaces with Ti-O Configurations: Evidence from X-ray Photoelectron Spectroscopy.

A chemical redox reaction can lead to a two-dimensional electron gas at the interface between a TiO2-terminated SrTiO3 (STO) substrate and an amorphous LaAlO3 capping layer. When replacing the STO substrate with rutile and anatase TiO2 substrates, considerable differences in the interfacial conduction are observed. On the basis of X-ray photoelectron spectroscopy (XPS) and transport measurements, we conclude that the interfacial conduction comes from redox reactions, and that the differences among the materials systems result mainly from variations in the activation energies for the diffusion of oxygen vacancies at substrate surfaces.

Giant Tunability of the Two-Dimensional Electron Gas at the Interface of γ-Al2O3/SrTiO3.

Two-dimensional electron gases (2DEGs) formed at the interface between two oxide insulators provide a rich platform for the next generation of electronic devices. However, their high carrier density makes it rather challenging to control the interface properties under a low electric field through a dielectric solid insulator, that is, in the configuration of conventional field-effect transistors. To surpass this long-standing limit, we used ionic liquids as the dielectric layer for electrostatic gating of oxide interfaces in an electric double layer transistor (EDLT) configuration. Herein, we reported giant tunability of the physical properties of 2DEGs at the spinel/perovskite interface of γ-Al2O3/SrTiO3 (GAO/STO). By modulating the carrier density thus the band filling with ionic-liquid gating, the system experiences a Lifshitz transition at a critical carrier density of 3.0 × 1013 cm-2, where a remarkably strong enhancement of Rashba spin-orbit interaction and an emergence of Kondo effect at low temperatures are observed. Moreover, as the carrier concentration depletes with decreasing gating voltage, the electron mobility is enhanced by more than 6 times in magnitude, leading to the observation of clear quantum oscillations. The great tunability of GAO/STO interface by EDLT gating not only shows promise for design of oxide devices with on-demand properties but also sheds new light on the electronic structure of 2DEG at the nonisostructural spinel/perovskite interface.

Alternatives to mineral oil adjuvants in vaccines against Aeromonas salmonicida subsp. salmonicida in rainbow trout offer reductions in adverse effects.

In an effort to reduce the frequency and severity of adverse reactions seen from the use of mineral oil adjuvants in salmonid fish, the effects of two alternative adjuvants were assessed, focusing on the induction of adverse effects as well as protection. Using rainbow trout (Oncorhynchus mykiss) as recipients, injection vaccines based on formalin-inactivated Aeromonas salmonicida subspecies salmonicida were formulated with CpG oligodeoxynucleotides, the liposomal cationic adjuvant formulation 01 (CAF01) or with Freund's incomplete adjuvant and administered intraperitoneally. Control groups of unvaccinated, Tris-buffered saline-injected or bacterin-injected individuals were included, and each group included in the study held a total number of 240 individuals. Subsequently, individuals from each group were examined for differences in Fulton's condition factor, macro- and microscopic pathological changes, as well as protection against experimental infection with A. salmonicida. While adverse effects were not eliminated, reductions in microscopic and macroscopic adverse effects, in particular, were seen for both the nucleotide- and liposome-based vaccine formulations. Furthermore, the induced protection appears similar to that of the benchmark formulation, thus introducing viable, potential alternative types of adjuvants for use in future fish vaccines.