Antigen Adjuvant Research Articles

Enhancement of Th1 immune response against Leishmania cysteine peptidase A, B by PLGA nanoparticle

Recombinant cysteine peptidase vaccine can induce protective immunity against cutaneous leishmaniasis. However, the antigenic diversity and variable immunogenicity prevents them from being approved for general vaccination. Different approaches like adjuvant application and antigen delivery systems are studied to increase their efficacy. Nanoparticles can both stimulated antigen uptakes and affect direction of immune response. In this study the effect of PLGA nanoparticles were considered to enhance the immune response against recombinant CPA (CPA) and CPB (CPB). For this purpose, L. major CPA and CPB were prepared. PLGA were conjugated to the proteins using Aldehyde/Hydrazine Reaction. Conjugation efficacy and created nanoparticle morphology were determined by FTIR and SEM methods, respectively. BALB/c mice were received intraperitoneally three boosts of 7 μg/mouse of each antigen alone (CPA/CPB/CPA + CPB) or as PLGA conjugated form in different Study groups, at 3 weeks interval. After vaccination, mice were challenged with 106L. major, subcutaneously. Time course study of lesion development demonstrated nanoparticle efficacy in parasite dissemination control that confirmed by spleen parasite burden assay. Significant induction of nitric oxide production by peritoneal macrophages and increase in splenocyte IFN-γ production showed the protective effect of PLGA-CPA/CPB vaccination in comparison to CPA and CPB alone. Current study demonstrated that the conjugation of the antigen with the PLGA can activate immune responses against L. major. However, further study is necessary to assess the long-term effect and other aspects of immune response.

Antigen-adjuvant effects of icariin in enhancing tumor-specific immunity in mastocytoma-bearing DBA/2J mice

Cancer immunotherapy has attracted much attention in recent years because of the ability of immune system to identify tumor cells and limit their growth. Icariin (ICA) is a natural flavonoid glucoside isolated from Epimedium plants and has shown a variety of pharmacological activities such as anti-inflammatory effects, immunological regulation and anticancer potency. Furthermore, it has immunoadjuvant effects on enhancing Th1-immune response, suggesting that ICA may serve as an adjuvant for cancer immunotherapy. In this study, we used P815 mouse mastocytoma tumor model and immunized them with P815AB peptide and/or ICA. Our results demonstrated that ICA could increase the cytotoxic T lymphocytes (CTL) response for P815AB peptide on the tumor-bearing DBA/2J mice. In addition, the percentage of CD4+CD8+/CD3+CD69+/CD69+NKG2D+ positive cells in splenocytes of the tumor-bearing mice all significantly increased after combined immunization with ICA and P815AB peptide. This illustrated that ICA could enhance the immunogenicity of P815AB and improve the ability of T cells and CTLs in recognizing the tumor cells. Moreover, ICA improved the function of peritoneal macrophages with effects of inhibition on tumor growth. Besides, we discussed the possible mechanism of ICA to enhance body immunity by detecting the expression level of MHC-I and related genes in B16-F10 and RMA/S cells. The results suggested that ICA has the potential to up-regulate LMP/TAP related molecules and induce the expression of MHC-I, which increase the immune surveillance and keep cancer in remission. In conclusion, ICA showed an anti-tumor effect both in vitro and in vivo and may be an effective antigen adjuvant for cancer treatment by enhancing tumor-specific immunity.

Experimental Autoimmune Encephalomyelitis (EAE) Model of Cynomolgus Macaques Induced by Recombinant Human MOG1-125 (rhMOG1-125) Protein and MOG34-56 Peptide.

Experimental autoimmune encephalomyelitis (EAE) induced by self-myelin antigen is a widely used in multiple sclerosis (MS) model for preclinical studies of new therapeutics and potential pathogenesis. By comparison with rodent EAE models, EAE models in primates are more similar to MS. Some groups have developed EAE models in primates by using common marmoset (Callithrix jacchus). However, this model has some limitations. EAE in cynomolgus monkey (Macaque fasciculrais) could overcome these limitations. The main objective of this study was to establish an ideal EAE cynomolgus monkey model resembling human MS by immunizing human myelin oligodendrocyte glycoprotein (MOG) protein. In this study, six female cynomolgus monkeys were divided into two separate experiment groups and one monkey as the control. EAE was induced in cynomolgus monkey by immunizing with the recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1-125) (rhMOG1-125) and a synthetic peptide, representing peptide 34-56 of human myelin oligodendrocyte glycoprotein (MOG34-56) in complete Freund's adjuvant (CFA) by subcutaneous multipoint immunization in the armpit and inguinal region and in combination with intravenous injection of pertussis toxin, and subsequent booster immunizations with the same dose of antigen in incomplete Freund's adjuvant (IFA) until the animals developed clinically significant EAE (score≥2). The body weight, clinical scores, magnetic resonance imaging (MRI), histopathology, antibody, cytokine profiling and antigen-specific lymphocyte proliferation were evaluated. The results showed that despite the different time intervals between onset and significant neurological deficits, all the cynomolgus monkeys immunized with rhMOG1-125 or MOG34-56 developed clinically significant acute fulminant or mild forms of EAE, with a success rate of 100%. The clinical courses were obvious heterogeneity which closely resembles MS. MOG34-56 immunization was much milder compared with rhMOG1-125 immunized individuals, at least in cynomolgus monkeys. Inflammation and demyelinated lesions were present in the brains and spinal cords. Immune profiling revealed high IgG levels associated with early onset of EAE but not the course of the disease. Significant antigen-specific T lymphocyte responses against immunodominant epitopes of MOG were also detected. Our results indicated rhMOG1-125 and MOG34-56 could induce successfully EAE models resembling MS in cynomolgus monkeys. The synergistic action of anti-myelin T cells and Abs during the pathogenesis of EAE could establish a more ideal EAE model.

Adjuvant Activity of Poly-ε-caprolactone/Chitosan Nanoparticles Characterized by Mast Cell Activation and IFN-γ and IL-17 Production.

Polymeric nanoparticles (NPs) are extremely attractive vaccine adjuvants, able to promote antigen delivery and in some instances, exert intrinsic immunostimulatory properties that enhance antigen specific humoral and cellular immune responses. The poly-ε-caprolactone (PCL)/chitosan NPs were designed with the aim of being able to combine the properties of the 2 polymers in the preparation of an adjuvant for the hepatitis B surface antigen (HBsAg). This article reports important results of an in vitro mechanistic study and immunization studies with HBsAg associated with different concentrations of the nanoparticles. The results revealed that PCL/chitosan NPs promoted mast cell (MC) activation (β-hexosaminidase release) and that its adjuvant effect is not mediated by the TNF-α secretion. Moreover, we demonstrated that HBsAg loaded PCL/chitosan NPs, administered through the subcutaneous (SC) route, were able to induce higher specific antibody titers without increasing IgE when compared to a commercial vaccine, and that the IgG titers are nanoparticle-dose dependent. The results also revealed the NPs' capability to promote a cellular immune response against HBsAg, characterized by the production of IFN-γ and IL-17. These results demonstrated that PCL/chitosan NPs are a good hepatitis B antigen adjuvant, with direct influence on the intensity and type of the immune response generated.

Engineering detoxified pneumococcal pneumolysin derivative ΔA146PLY for self-biomineralization of calcium phosphate: Assessment of their protective efficacy in murine infection models

Vaccine design ushered in the era of nanotechnology, as the vaccine is being developed toward particulate formulation. We have previously shown that the attenuated pneumolysin mutant (ΔA146PLY) was a safe and effective pneumococcal vaccine candidate. Here, to further optimize the formulation, we fused calcium phosphate (CaP) binding domains with ΔA146PLY so that the biocompatible CaP can mineralize with the protein automatically, allowing simple production of nanoparticle antigen during preparation. We fabricated four different nanoparticles, and then we compared the characteristics of different CaP-ΔA146PLY nanoparticles and demonstrated the influence of CaP binding domains on the size, shape and surface calcium content of the nanoparticles. It was found that these self-biomineralized CaP-ΔA146PLY nanoparticles varied in their capacity to induce BMDCs and splenocytes production of cytokines. We further demonstrated that, compared to free proteins, nanoparticle antigens induced more efficient humoral and cellular immune responses which was strong enough to protect mice from both pneumonia and sepsis infection. Also, the integration of CaP to protein has no significant impairment on body weight of animals, and subcutaneous injection of ΔA146PLY-peptides@CaP nanoparticles did not lead to permanent formation of nodules in the skin relative to Alum adjuvant formulated antigens. Together, our data sufficiently suggest that soluble ΔA146PLY vaccine candidate could be processed into nanoparticles by self-biomineralization of CaP, the immunogenicity of which could be efficiently improved by the CaP binding domains and biomineralization.

Liposomes-coated gold nanocages with antigens and adjuvants targeted delivery to dendritic cells for enhancing antitumor immune response

For nanovaccine-based cancer immunotherapy, dendritic cells (DCs) are one of the most powerful antigen presenting cells (APCs) that initiate and promote the maturation of antigen-specific cytotoxic T lymphocytes (e.g., CD8+ T cells) to induce the local and systemic antitumor immunity and further suppress the tumor metastasis and produce long-term protection against tumor. Thus, the activation and maturation of DCs is the prerequisite for efficient CD8+ T cell-based antitumor immune responses, which is considered as a primary and promising task for nanovaccine engineering. Herein, we introduce a versatile nanovaccine of liposomes-coated gold nanocages (Lipos-AuNCs) modified with DCs specific antibody aCD11c for targeted delivery of adjuvant MPLA and melanoma antigen peptide TRP2 to promote the activation and maturation of DCs, and enhance tumor specific T lymphocytes responses. Moreover, AuNCs accumulation and AuNCs-engulfed DCs migration to regional lymph nodes (RLNs) became real-time visualization through in vivo fluorescence and photoacoustic (PA) imaging to monitor the immunity process. In vivo experimental results demonstrated that the targeted antigen/adjuvants-loaded AuNCs exhibited enhanced antitumor immune response to inhibit tumor growth and metastasis in both B16-F10 prophylactic and lung metastasis models, which may act as a promising nanoplatform for antitumor immunotherapy and in vivo tracking.

What Have We Learnt about BCG Vaccination in the Last 20 Years?

A number of new tuberculosis (TB) vaccines have been or are entering clinical trials, which include genetically modified mycobacteria, mycobacterial antigens delivered by viral vectors, or mycobacterial antigens in adjuvant. Some of these vaccines aim to replace the existing BCG vaccine but others will be given as a boosting vaccine following BCG vaccination given soon after birth. It is clear that the existing BCG vaccines provide incomplete and variable protection against pulmonary TB. This review will discuss what we have learnt over the last 20 years about how the BCG vaccine induces specific and non-specific immunity, what factors influence the immune responses induced by BCG, and progress toward identifying correlates of immunity against TB from BCG vaccination studies. There is still a lot to learn about the BCG vaccine and the insights gained can help the development of more protective vaccines.

Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands Work Additively via MyD88 To Induce Protective Antiviral Immunity in Mice.

We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen induction of humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Modulation of Immunogenicity and Conformation of HA1 Subunit of Influenza A Virus H1/N1 Hemagglutinin in Tubular Immunostimulating Complexes.

The HA1 subunit of the influenza virus hemagglutinin (HA) is a valuable antigen for the development of vaccines against flu due to the availability of most antigenic sites which are conformational. Therefore, a novel adjuvanted antigen delivery system, tubular immunostimulating complexes (TI-complexes) comprising monogalactosyldiacylglycerol (MGDG) from different marine macrophytes as a lipid matrix for an antigen, was applied to enhance the immunogenicity of recombinant HA1 of influenza A H1N1 and to study the relation between its immunogenicity and conformation. The content of anti-HA1 antibodies and cytokines was estimated by ELISA after the immunization of mice with HA1 alone, and HA1 was incorporated in TI-complexes based on different MGDGs isolated from green algae Ulva lactuca, brown algae Sargassum pallidum, and seagrass Zostera marina. Conformational changes of HA1 were estimated by differential scanning calorimetry and intrinsic fluorescence. It was shown that the adjuvant activity of TI-complexes depends on the microviscosity of MGDGs, which differently influence the conformation of HA1. The highest production of anti-HA1 antibodies (compared with the control) was induced by HA1 incorporated in a TI-complex based on MGDG from S. pallidum, which provided the relaxation of the spatial structure and, likely, the proper presentation of the antigen to immunocompetent cells.

Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50°C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45°C and only a 10% loss was observed after 6monthsat 50°C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination.

Inflammatory monocytes regulate Th1 oriented immunity to CpG adjuvanted protein vaccines through production of IL-12

Due to their capacity to skew T cell responses towards Th1 oriented immunity, oligonucleotides containing unmethylated CpG motifs (CpG) have emerged as interesting adjuvants for vaccination. Whereas the signalling pathways in response to CpG mediated TLR9 activation have been extensively documented at the level of the individual cell, little is however known on the precise identity of the innate immune cells that govern T cell priming and polarisation to CpG adjuvanted protein antigens in vivo. In this study, we demonstrate that optimal induction of Th1 oriented immunity to CpG adjuvanted protein vaccines requires the coordinated actions of conventional DCs and of monocytes. Whilst conventional DCs were required for antigen presentation and initial T cell priming, monocytes constitute the main source of the Th1 polarising cytokine IL-12.

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund's adjuvant. A second immunization with the same antigens in incomplete Freund's adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund's adjuvant. A second immunization with the same antigens in incomplete Freund's adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses

BackgroundNewborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization.ObjectiveSmall-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs.MethodsChemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist–encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow–derived DCs enabled benchmarking of the TLR8 agonist–encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25–loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice.ResultsAlthough alum-adjuvanted vaccines induced modest costimulatory molecule expression, limited TH-polarizing cytokine production, and significant cell death, BCG induced a robust adult-like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL-12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist–adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B-specific CD4+ T-cell numbers.ConclusionTLR8 agonist–encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines.

Intravaginal immunisation using a novel antigen-releasing ring device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses

The generation of effective levels of antigen-specific immunity at the mucosal sites of pathogen entry is a key goal for vaccinologists. We explored topical vaginal application as an approach to initiate local antigen-specific immunity, enhance previously existing systemic immunity or re-target responses to the mucosae. To deliver a protein vaccine formulation to the vaginal mucosal surface, we used a novel vaginal ring device comprising a silicone elastomer body into which three freeze-dried, rod-shaped, hydroxypropylmethylcellulose inserts were incorporated. Each rod contained recombinant HIV-1 CN54gp140 protein (167μg)±R848 (167μg) adjuvant. The inserts were loaded into cavities within each ring such that only the ends of the inserts were initially exposed.Sheep received a prime-boost vaccination regime comprising intramuscular injection of 100μg CN54gp140+200μg R848 followed by three successive ring applications of one week duration and separated by one month intervals. Other sheep received only the ring devices without intramuscular priming. Serum and vaginal mucosal fluids were sampled every two weeks and analysed by CN54gp140 ELISA and antigen-specific B cells were measured by flow cytometry at necropsy. Vaccine antigen-specific serum antibody responses were detected in both the intramuscularly-primed and vaginal mucosally-primed groups. Those animals that received only vaginal vaccinations had identical IgG but superior IgA responses. Analysis revealed that all animals exhibited mucosal antigen-specific IgG and IgA with the IgA responses 30-fold greater than systemic levels. Importantly, very high numbers of antigen-specific B cells were detected in local genital draining lymph nodes.We have elicited local genital antigen-specific immune responses after topical application of an adjuvanted antigen formulation within a novel vaginal ring vaccine release device. This regimen and delivery method elicited high levels of antigen-specific mucosal IgA and large numbers of local antigen-reactive B cells, both likely essential for effective mucosal protection.

Abstract B002: TLR7/8-matured dendritic cells for therapeutic vaccination in AML: Results of a clinical Phase I/II trial

Abstract Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. Using a TLR7/8 agonist, we have developed a GMP-compliant 3-day protocol to differentiate monocytes of intensively pretreated AML patients into highly functional, therapeutic DCs. A phase I/II proof-of-concept study has been initiated using TLR7/8-matured DCs as postremission therapy of AML patients with a non-favorable risk profile in CR or CRi after intensive induction therapy (NCT01734304). DCs have been loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients have been vaccinated intradermally with 5×106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. In total, 13 patients have been enrolled into the study. The first 6 patients were analysed in phase I for safety and toxicity of the DC vaccine. No higher grade toxicities were observed during their treatment and hence phase II has been initiated. DCs of sufficient number and quality were generated from leukapheresis in 10/11 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 vaccinated patients, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by NGS revealed an enrichment of particular clonotypes at DTH sites. In addition, we detected vaccine-specific T-cell responses by multimer staining and by Interferon-gamma-ELISPOT analysis: 7/7 patients showed responses to CMVpp65 and 2/7 exhibited responses to PRAME and WT1, respectively. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial. We conclude that vaccination with TLR7/8-matured, LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. Citation Format: Katrin Deiser, Felix S. Lichtenegger, Frauke M. Schnorfeil, Thomas Köhnke, Torben Altmann, Veit Bücklein, Christian Augsberger, Andreas Moosmann, Monika Brüggemann, Mirjam HM Heemskerk, Beate Wagner, Wolfgang Hiddemann, Iris Bigalke, Gunnar Kvalheim, Marion Subklewe. TLR7/8-matured dendritic cells for therapeutic vaccination in AML: Results of a clinical Phase I/II trial [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B002.

Supramolecular peptide hydrogel adjuvanted subunit vaccine elicits protective antibody responses against West Nile virus

A crucial issue in vaccine development is to balance safety with immunogenicity. The low immunogenicity of most subunit antigens warrants a search for adjuvants able to stimulate both cell-mediated and humoral immunity. In recent years, successful applications of nanotechnology and bioengineering in the field of vaccine development have enabled the production of novel adjuvant technologies. In this work, we investigated totally synthetic and supramolecular peptide hydrogels as novel vaccine adjuvants in conjunction with the immunoprotective envelope protein domain III (EIII) of West Nile virus as an immunogen in a mouse model. Our results indicate that, compared to the clinically approved adjuvant alum, peptide hydrogel adjuvanted antigen elicited stronger antibody responses and conferred significant protection against mortality after virus challenge. The high chemical definition and biocompatibility of self-assembling peptide hydrogels makes them attractive as immune adjuvants for the production of subunit vaccines against viral and bacterial infections where antibody-mediated protection is desirable.

Behavioral abnormalities in female mice following administration of aluminum adjuvants and the human papillomavirus (HPV) vaccine Gardasil.

Vaccine adjuvants and vaccines may induce autoimmune and inflammatory manifestations in susceptible individuals. To date most human vaccine trials utilize aluminum (Al) adjuvants as placebos despite much evidence showing that Al in vaccine-relevant exposures can be toxic to humans and animals. We sought to evaluate the effects of Al adjuvant and the HPV vaccine Gardasil versus the true placebo on behavioral and inflammatory parameters in female mice. Six-week-old C57BL/6 female mice were injected with either, Gardasil, Gardasil+pertussis toxin (Pt), Al hydroxide, or, vehicle control in amounts equivalent to human exposure. At 7.5months of age, Gardasil and Al-injected mice spent significantly more time floating in the forced swimming test (FST) in comparison with vehicle-injected mice (Al, p=0.009; Gardasil, p=0.025; Gardasil+Pt, p=0.005). The increase in floating time was already highly significant at 4.5months of age for the Gardasil and Gardasil+Pt group (p≤0.0001). No significant differences were observed in the number of stairs climbed in the staircase test which measures locomotor activity. These results indicate that differences observed in the FST were unlikely due to locomotor dysfunction, but rather due to depression. Moreover, anti-HPV antibodies from the sera of Gardasil and Gardasil+Pt-injected mice showed cross-reactivity with the mouse brain protein extract. Immunohistochemistry analysis revealed microglial activation in the CA1 area of the hippocampus of Gardasil-injected mice. It appears that Gardasil via its Al adjuvant and HPV antigens has the ability to trigger neuroinflammation and autoimmune reactions, further leading to behavioral changes.

Preclinical safety study of a recombinant Streptococcus pyogenes vaccine formulated with aluminum adjuvant.

A recombinant vaccine composed of a fusion protein formulated with aluminum hydroxide adjuvant is under development for protection against diseases caused by Streptococcus pyogenes. The safety and local reactogenicity of the vaccine was assessed by a comprehensive series of clinical, pathologic and immunologic tests in preclinical experiments. Outbred mice received three intramuscular injections of 1/5th of the human dose (0.1 ml) and rabbits received two injections of the full human dose. Control groups received adjuvant or protein antigen. The vaccine did not cause clinical evidence of systemic toxicity in mice or rabbits. There was a transient increase of peripheral blood neutrophils after the third vaccination of mice. In addition, the concentration of acute phase proteins serum amyloid A and haptoglobin was significantly increased 1 day after injection of the vaccine in mice. There was mild transient swelling and erythema of the injection site in both mice and rabbits. Treatment-related pathology was limited to inflammation at the injection site and accumulation of adjuvant-containing macrophages in the draining lymph nodes. In conclusion, the absence of clinical toxicity in two animal species suggest that the vaccine is safe for use in a phase I human clinical trial. Copyright © 2016 John Wiley & Sons, Ltd.

The immunotherapeutic GEN-003 as a prophylactic vaccine candidate for genital herpes in guinea pigs

Abstract GEN‑003 is a subunit vaccine candidate containing adjuvanted gD and ICP4 antigens that has shown durable therapeutic efficacy against genital herpes in a phase 2 clinical trial. Here we describe the potential benefit of GEN-003 as a prophylactic vaccine in a guinea pig model of genital herpes. Animals were immunized three times every two weeks with GEN-003 or control and intravaginally challenged with herpes simplex virus 2 (HSV-2, MS strain) 21 days later. GEN-003 significantly reduced frequency and severity of genital lesions in both acute and early stages of recurrent infection by over 85%, and 60% of vaccinated guinea pigs did not develop any lesions during the 33 day follow-up period. Furthermore, viral genome copy numbers recovered in vaginal swabs from GEN-003-immunized animals were 100-fold lower than from controls. Recurrent shedding frequencies were reduced by 27%. GEN-003 vaccination induced antigen-specific antibody responses at least as strong as those elicited by viral infection. Driven by gD-specific antibodies, HSV-2 neutralizing antibody titers followed the same pattern. After viral challenge, peripheral antigen-specific T cell responses in spleens of GEN-003-immunized animals were lower than those in controls, as measured by interferon-γ ELISpot. This might have resulted from increased antigen-specific T cell recruitment to the genital mucosa, or decreased exposure to the virus due to fewer recurrences. Additional methods to better understand mucosal mechanisms of protection are in development. In summary, GEN-003 shows promise as a prophylactic vaccine candidate for genital herpes. Ongoing studies will determine if protection can be further enhanced by addition or exchange of antigens and/or adjuvants.