OVA-specific Antibody Responses Research Articles

Differential Regulation of DC Function, Adaptive Immunity, and MyD88 Dependence by MF59 and AS03-like Adjuvants.

MF59 and AS03 are squalene emulsion-based vaccine adjuvants with similar compositions and droplet sizes. Despite their broad use in licensed influenza vaccines, few studies compared their adjuvant effects and action mechanisms side by side. Considering the majority of adjuvants act on dendritic cells (DCs) to achieve their adjuvant effects, this study compared MF59 and AS03-like adjuvants (AddaVax and AddaS03, respectively) to enhance antigen uptake, DC maturation, ovalbumin (OVA) and seasonal influenza vaccine-induced immune responses. Considering MF59 was reported to activate MyD88 to mediate its adjuvant effects, this study also investigated whether the above-explored adjuvant effects of AddaVax and AddaS03 depended on MyD88. We found AddaVax more potently enhanced antigen uptake at the local injection site, while AddaS03 more potently enhanced antigen uptake in the draining lymph nodes. AddaS03 but not AddaVax stimulated DC maturation. Adjuvant-enhanced antigen uptake was MyD88 independent, while AddaS03-induced DC maturation was MyD88 dependent. AddaVax and AddaS03 similarly enhanced OVA-induced IgG and subtype IgG1 antibody responses as well as influenza vaccine-induced hemagglutination inhibition antibody titers, whileAddaS03 more potently enhanced OVA-specific IgG2c antibody responses. Both adjuvants depended on MyD88 to enhance vaccine-induced antibody responses, while AddaVax depended more on MyD88 to achieve its adjuvant effects. Our study reveals similarities and differences of the two squalene emulsion-based vaccine adjuvants, contributing to our improved understanding of their action mechanisms.

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice.

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.

Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01)

Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen-specific CD8+ T-cell and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen-specific humoral immune responses against subunit protein antigens.

Lentinan-functionalized graphene oxide hydrogel as a sustained antigen delivery system for vaccines

Hydrogel has been proven to have the ability to deliver antigens continuously to achieve slow vaccine delivery, which makes it a promising candidate for an adjuvant delivery platform. Meanwhile, graphene oxide (GO) has garnered significant attention due to its good biosafety, excellent surface area and easy modification. However, GO exists as weak colloidal particles and poses challenges in self-assembling into a hydrogel structure. Here, we propose an innovative strategy involving self-assembling lentinan-functionalized graphene oxide hydrogel ((LNT-GO Gel) by simply mixing lentinan (LNT)-functionalized GO with polyethylene imide (PEI), which can simultaneously encapsulate antigens, achieve long-lasting release of antigens and generate excellent adjuvant activity. The results indicated that the LNT-GO Gel can control the release of OVA at the injection site and confer targeted delivering capacity to lymph nodes. And the date demonstrates that LNT-GO Gel displays favorable safety and biodegradability in vivo. Moreover, LNT-GO Gel can enhance the activation and maturation of dendritic cells (DCs) in lymph node, induce stronger OVA-specific antibody response, and promote spleen T lymphocyte differentiation, which underscores that LNT-GO Gel has ability to generate stronger antigen-specific humoral and cellular immune responses. Collectively, these results demonstrate the adjuvant potential of the lentinan-functionalized graphene oxide hydrogel (LNT-GO Gel) for subunit vaccine.

Heterologous prime-boost vaccine using antigen-loaded microparticles and adenovirus (encoding antigen) enhances cellular immune responses and antitumor activity

Heterologous prime-boost vaccines have the potential to promote higher immune responses than homologous prime-boost vaccines and were used in this murine study to investigate the effect on the magnitude of the cellular (and humoral) antigen-specific immune responses and antitumor efficacy when a microparticle formulation (prime) is combined with an adenoviral vaccine (boost). Specifically, the prime comprised chick egg ovalbumin (OVA; 25 µg/dose), used here as a model tumor antigen (TA), encapsulated in microparticles (∼700 nm diameter) made from the biodegradable polymer, 50:50 poly(lactic-co-glycolic acid) (PLGA); while attenuated adenovirus (type 5) encoding OVA (Ad5OVA; 108 PFU/dose) was employed as the boost. The ability of OVA-loaded microparticles to enhance OVA-specific antibody responses, OVA-specific CD3 + CD8 + T cell responses and antitumor activity (i.e., protection against OVA-expressing tumor-challenge) to the heterologous prime-boost vaccine was investigated; and it was found that this prime-boost combination could significantly enhance OVA-specific cellular responses compared to all other vaccination groups and was the only group to confer a significant survival advantage over the unvaccinated group (naïve) in a prophylactic animal tumor model. This finding illustrates the potential for combining TA-loaded PLGA-based microparticles with other vaccine formats to improve tumor-specific cellular immune responses.

Astragalus polysaccharides combined with simvastatin as an immunostimulant enhances the immune adjuvanticity of oil-in-water emulsion and immune responses in mice.

Oil-in-water emulsion-based adjuvants have demonstrated acceptable safety in many disease indications, while their adjuvant activities for vaccines still need to be improved. Recently, the strategy of combining adjuvants with multiple types of immunostimulants has been shown to enhance immune responses. In this study, astragalus polysaccharides were combined with simvastatin as an immunostimulant to construct a compound O/W emulsion adjuvant. The formulations were optimized according to the OVA-specific antibody responses induced in mice. For this reason, high (5mg/mL), medium (2.5mg/mL), and low (1.25mg/mL) concentrations of astragalus polysaccharides and high (10mg/mL), medium (1mg/mL), and low (0.1mg/mL) concentrations of simvastatin were selected. The final optimal formulation of the immunostimulant was a high concentration of astragalus polysaccharides combined with a medium concentration of simvastatin. The optimal compound O/W emulsion adjuvant could induce effective humoral and cellular immune responses that were stronger and more stable than those induced by aluminum adjuvant and Freund's adjuvant. The OVA/HAPS-MSim-OE induced dramatically strong and persistent IgG expressions and Th1-polarized immune responses. What's more, the highest CD4+/CD8+lymphocyte ratios were observed in OVA/HAPS-MSim-OE group. In addition, compound O/W emulsion adjuvant groups significantly promoted the secretion of IFN-γ and IL-6, which also indicated that the compound O/W emulsion adjuvants could induce both enhanced Th1 and Th2-mediated immune responses but prefer the Th1-mediated ones. This study would contribute to an interesting and promising direction in the development of emulsion-based adjuvants.

R-DOTAP (Versamune): A novel enantiospecific cationic lipid nanoparticle that induces CD4 and CD8 cellular immune responses to whole protein and tumor-specific peptide antigens.

e15211 Background: Immunotherapy approaches are limited in their ability to induce antigen-specific CD8+ T cells in vivo able to recognize and kill tumor cells. We developed a novel immunotherapy approach using enantiomerically pure, R-DOTAP cationic lipid nanoparticles and tumor-derived T cell antigens, and previously demonstrated that R-DOTAP formulations efficiently prime cytotoxic T cells through enhanced cross presentation and induction of type I interferons.[1] A phase I clinical trial of a R-DOTAP HPV16 peptide formulation confirmed induction of strong in vivo HPV-specific CD8+ cytolytic T-cells without associated systemic toxicities. In this study, we assessed R-DOTAP nanoparticle formulations containing whole protein (ovalbumin) or long multi-epitope peptides from the tumor antigen TARP (T-cell alternate reading frame protein): a 58-residue protein overexpressed in prostate and breast cancers, documented to be immunogenic in humans. Methods: R-DOTAP formulations were prepared containing ovalbumin (OVA) or TARP peptides. C57BL/6K mice were immunized with 10 μg/mouse of OVA plus R-DOTAP, CFA or sucrose on Days 0, 15 and 30. OVA-specific cellular and humoral responses following vaccination were assessed by measuring splenic CD4 and CD8 T cell IFN-γ production and circulating OVA-specific antibodies in serum. HLA-A2 transgenic mice (AAD mice) were vaccinated with long, multi-epitope TARP peptides delivered as an R-DOTAP admixture or with CFA or sucrose on Days 0 and 7. Antigen-specific T cell responses were measured by IFN-γ ELISpot assay. Results: OVA R-DOTAP formulations induced strong antigen-specific effector CD4 and CD8 immune and memory responses detected 7 and 30 days, respectively, following vaccination as well as OVA-specific antibody responses. In TARP peptide vaccinated mice, R-DOTAP formulations were able to present multiple CD8 T cell epitopes and stimulate responses that were superior to CFA. Conclusions: Our results suggest that R-DOTAP is a versatile immune activating therapy that can be formulated with long, multi-epitope tumor-derived peptides or whole proteins. R-DOTAP formulations induce quantitatively robust antigen-specific CD4 and CD8 T cells in vivo compared to well-established immune stimulants. Reference: 1.Gandhapudi SK, Ward M, Bush JP et al. Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response. J Immunol 2019;202:3524-3536

Single-injecting, bioinspired nanocomposite hydrogel that can recruit host immune cells in situ to elicit potent and long-lasting humoral immune responses

Vaccination is an effective medical intervention for preventing disease. However, without an adjuvant, most subunit vaccines are poorly immunogenic. This work develops a bioinspired nanocomposite hyaluronic acid hydrogel system that incorporates N-trimethyl chitosan nanoparticles (TMC/NPs) that carry a model subunit vaccine ovalbumin (OVA) that can elicit a potent and prolonged antigen-specific humoral response. Experimental results indicate that the nanocomposite hydrogel system (NPs-Gel) can retain a large proportion of its TMC/NPs that are bonded by covalent/electrostatic interactions and extend the release of the encapsulated OVA, enabling their localization at the site of hydrogel injection. The positively charged TMC/NPs can be effectively internalized by dendritic cells, significantly augmenting their maturation, suggesting that TMC can function as an adjuvant-based OVA delivery system. Upon subcutaneous implantation in mice, the NPs-Gel acts as an in situ depot that recruits and concentrates immune cells. The TMC/NPs that do not have any specific interactions with the hydrogel network are released rapidly and internalized by the neighboring immune cells, providing a priming dose, while those retained inside the NPs-Gel are ingested by the recruited and concentrated immune cells over time, acting as a booster dose, eliciting high titers of OVA-specific antibody responses. These experimental results suggest particulate vaccines that are integrated in such a bioinspired hydrogel system may be used as single-injection prime-boost vaccines, enabling effective and persistent humoral immune responses.

Experimental protocol for development of adjuvant-free murine chronic model of allergic asthma

BackgroundMouse models of allergic asthma play a crucial role in exploring of asthma pathogenesis and testing of novel anti-inflammatory drugs. Widely used acute asthma models usually developed with adjuvant (aluminum hydroxide (alum)) do not reproduce one of the main asthma feature - airway remodeling while chronic asthma model mimic the pathophysiology of human disease. Moreover, the use of alum causes distress in experimental animals and impedes the test of adjuvant-containing drugs. In this study, we aimed to develop a chronic adjuvant-free asthma model with pronounced asthmatic phenotype. MethodsFemale BALB/c mice were divided into 3 groups. The first group was sensitized with intraperitoneal injections of ovalbumin (OVA) emulsified in aluminum hydroxide on days 0, 14, 28 followed by two stages of intranasally challenge with OVA on days 41–43 and 62–64. The second group was subcutaneously sensitized with the same dose of OVA without adjuvant and challenged on the same days. The third group (negative control) included mice which did not received any kind of treatment (i.e. sensitization and challenge). Serum levels of OVA-specific IgE, IgG2a and IgG1 antibodies were detected by ELISA. Airway hyper-responsiveness was measured by non-invasive plethysmography on days 44 and 65. Bronchoalveolar lavage fluids (BALF) sampled in all groups on days 45 and 66 were analyzed by light microscopy. The left lung was removed for histological analysis. The IL-4 and IFNγ mRNA expression in BALF cells was evaluated by RT-PCR. ResultsThe OVA-specific IgE antibody response was two-fold increased in mice from adjuvant-free group compared to the adjuvant group that reflects reorientation of immune response towards Th2 phenotype. At the same time, the level of OVA-specific IgG1 and IgG2a antibodies was increased in the adjuvant group. Airway hyperresponsiveness to methacholine in mice of both experimental groups was two-fold higher than in control. Analysis of cell composition in BAL has shown a significant increase in eosinophil count in both experimental groups that indicate the development of allergic inflammation. Lung histology revealed airway remodeling in both experimental groups including goblet cell hyperplasia/metaplasia, thickening of airway walls, collagen deposition in the wall of distal airways. Additionally, the tendency to develop hypertrophy of bronchial smooth muscle layer was observed. Study of gene expression in BAL cells revealed the increase of IL-4 level in both adjuvant and adjuvant-free groups while IFNγ expression in both experimental groups was similar to control group. ConclusionWe have developed a chronic adjuvant-free mouse asthma model which possesses all necessary features of the disease including airway remodeling and is more suitable for pre-clinical evaluation of novel therapeutic approaches including adjuvant-containing drugs.

Mouse DC-SIGN/CD209a as Target for Antigen Delivery and Adaptive Immunity.

The efficacy of vaccination studies aimed at targeting antigens to human DC-SIGN (hDC-SIGN) have been notoriously difficult to study in vivo, as eight dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) homologs have been described in mice. CD209a/SIGNR5 has been coined as the mouse DC-SIGN (mDC-SIGN) ortholog, based on its expression and location in the genome. Nonetheless, which properties of hDC-SIGN are covered by mDC-SIGN is poorly investigated. One of the most important functions of DC-SIGN is the induction of adaptive immunity. As such, the aim of this study is to determine the capability of mDC-SIGN to induce adaptive immune responses. Here, we show that mDC-SIGN is expressed on GM-CSF cultured bone marrow-derived dendritic cells (BMDCs) and macrophages. However, mDC-SIGN is an internalizing receptor which, unlike hDC-SIGN, quickly resurfaces after internalization. Binding of OVA-coupled anti-mDC-SIGN antibody by BMDCs leads to quick internalization, processing, and presentation to antigen-specific CD8+ and CD4+ T cells, which can be boosted using the TLR4 ligand, monophosphoryl lipid A. In the homeostatic condition, mDC-SIGN is mostly expressed on myeloid cells in the skin and spleen. A subcutaneous injection of fluorescent anti-mDC-SIGN reveals specific targeting to mDC-SIGN+ skin dendritic cells (DCs) and monocyte-derived DCs in situ. A subcutaneous vaccination strategy containing OVA-coupled anti-mDC-SIGN antibody generated antigen-specific polyfunctional CD8+ T cell and CD4+ T cell responses and a strong isotype-switched OVA-specific antibody response in vivo. We conclude that mDC-SIGN shows partly overlapping similarities to hDC-SIGN and that targeting mDC-SIGN provides a valuable approach to investigate the immunological function of DC-SIGN in vivo.

Humoral Immune Response Modulation by Aryl hydrocarbon Receptor Activation (AhR) in CD11c+ Cells: Exploring Gender-specific and Ligand-dependent Effects on Antibody Responses in Mice.

Abstract Modulation of antibody responses is often a summation of effects on B cells, helper T cells, and dendritic cells (DCs). All three of these immune cells express AhR, a ligand-activated transcription factor with endogenous and exogenous ligands known to modulate immune responses. Importantly, antibody responses are keenly sensitive to AhR activation by TCDD, its prototypical ligand and common environmental pollutant. However, the specific contribution of AhR activation in DCs has not yet been defined, particularly in the context of gender-specific and ligand-dependent effects. We aimed to determine the effects of AhR activation in DCs by endogenous and exogenous ligands on OVA-specific antibody responses in mice. Specifically, we hypothesized that AhR activation in DCs alone suppresses antigen-specific serum antibody production, selectively affecting different isotypes. To test this hypothesis, we used mice conditionally deficient of AhR in CD11c+ DCs (AHRCD11c). Control (AhRd) and AHRCD11c mice were treated with vehicle, ITE or TCDD via oral gavage, and subsequently immunized i.p. with whole OVA/adjuvant to induce a systemic, antigen-specific humoral response. One week later, AhRd mice showed significantly reduced OVA-specific IgM and IgG2a titers while IgA was significantly increased; effects that were ameliorated in AhRCD11c mice. Interestingly, TCDD-induced IgA increases were both AhR- and gender-dependent with female mice showing increased IgA production. Collectively, these results demonstrate that DCs contribute to, but are not wholly responsible for, AhR-mediated effects on antigen-specific serum antibody generation. Research supported by ES013784 (DMS) and AAI Careers in Immunology Fellowship (JMK).

The effect of the common food additive tBHQ in OVA-elicited food allergy

Abstract Immune-mediated adverse reactions to food allergens are rising at an alarming rate on a global scale for reasons that are not completely understood. Although many factors such as microbiota, diet, obesity, and environmental chemical exposure have been proposed to contribute to this marked increase, the identification of specific causative factors has remained elusive. Here we tested the hypothesis that a common food additive, tert-butylhydroquinone (tBHQ), at concentrations relevant to human exposure, exacerbates allergic response in ovalbumin (OVA) sensitized mice. Female Balb/c mice (4 weeks old) were maintained on control diet or diet with 0.001% tBHQ for the duration of these studies. The mice were exposed to OVA once per week for 4 weeks during the sensitization phase. Sensitization to OVA was assessed by the rise in OVA-specific IgE and IgG1. Upon oral challenge, mice were monitored for hypothermia shock response (HSR) and mast cell protease (mMCP)-1 response. Although sensitization with OVA elicited a robust OVA-specific IgE antibody response in both the control and tBHQ diet groups, IgE levels were markedly higher in the mice on the tBHQ diet as compared to control diet. Likewise, in response to OVA challenge, a more marked decrease in body temperature was observed in the mice on the tBHQ diet. Furthermore, the mMCP-1 response to OVA challenge was 3-fold greater in the mice on the tBHQ diet as compared to control diet. Taken together, we report low concentrations of the food additive tBHQ promote OVA sensitization and exacerbate anaphylactic response to OVA challenge in a mouse model of food allergy.

Evaluation of pH-sensitive fusogenic polymer-modified liposomes co-loaded with antigen and α-galactosylceramide as an anti-tumor vaccine.

pH-Sensitive fusogenic polymer-modified (pH-sensitive) liposomes co-loaded with tumor model antigen, ovalbumin (OVA), and adjuvant, α-galactosylceramide (α-GalCer) were fabricated and administered subcutaneously into mice. The ability of pH-sensitive liposomes containing OVA and α-GalCer to stimulate cellular and humoral immune responses in vivo was compared with OVA-encapsulating pH-sensitive liposomes as well as with OVA alone. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, antigen-specific IgG1 antibody responses were noted in mice immunized with OVA alone, whereas immunization with OVA-containing pH-sensitive liposomes and with pH-sensitive liposomes containing OVA and α-GalCer resulted in the induction of OVA-specific IgG1 and IgG2b antibody responses. Moreover, more substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from mice immunized with pH-sensitive liposomes having OVA and α-GalCer than OVA-containing pH-sensitive liposomes in vitro. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA tumor cells. In addition, prophylactic vaccination efficacy against tumor formation was evaluated. In all mice immunized with pH-sensitive liposomes having OVA and α-GalCer, immunization provided substantial protection from tumor formation. The therapeutic efficacy of pH-sensitive liposomes containing OVA and α-GalCer against already established E.G7-OVA tumors was also investigated. Tumor growth was reduced significantly in all mice treated with pH-sensitive liposomes having OVA and α-GalCer. The provided evidence on the advantage of antigen and α-GalCer co-encapsulation into pH-sensitive liposomes should be considered in the design of future cancer vaccines for prophylactic and therapeutic purposes.

Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?

Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an in vivo ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response.

Aluminum (Oxy)Hydroxide Nanosticks Synthesized in Bicontinuous Reverse Microemulsion Have Potent Vaccine Adjuvant Activity.

Insoluble aluminum salts such as aluminum (oxy)hydroxide are commonly used as vaccine adjuvants. Recently, there is evidence suggesting that the adjuvant activity of aluminum salt-based materials is tightly related to their physicochemical properties, including nanometer-scale size, shape with long aspect ratio, and low degree of crystallinity. Herein, for the first time, the bicontinuous reverse microemulsion (RM) technique was utilized to synthesize stick-like monodisperse aluminum (oxy)hydroxide nanoparticles with a long aspect ratio of ∼10, length of ∼80 nm, and low degree of crystallinity (denoted as Al-nanosticks). Moreover, the relationship between the physicochemical properties of Al-nanosticks and the bicontinuous RM was discussed. Compared to the commercial Alhydrogel, which contains micrometer-scale aluminum oxyhydroxide particular aggregates with moderate degree of crystallinity, the Al-nanosticks are more effective in adsorbing and delivering antigens (e.g., ovalbumin, OVA) into antigen-presenting cells, activating inflammasomes, and potentiating OVA-specific antibody responses in a mouse model. It is concluded that the aluminum (oxy)hydroxide nanosticks synthesized in the bicontinuous RM are promising new aluminum salt-based vaccine adjuvants.

Graphene Oxides Decorated with Carnosine as an Adjuvant To Modulate Innate Immune and Improve Adaptive Immunity in Vivo.

Current studies have revealed the immune effects of graphene oxide (GO) and have utilized them as vaccine carriers and adjuvants. However, GO easily induces strong oxidative stress and inflammatory reaction at the site of injection. It is very necessary to develop an alternative adjuvant based on graphene oxide derivatives for improving immune responses and decreasing side effects. Carnosine (Car) is an outstanding and safe antioxidant. Herein, the feasibility and efficiency of ultrasmall graphene oxide decorated with carnosine as an alternative immune adjuvant were explored. OVA@GO-Car was prepared by simply mixing ovalbumin (OVA, a model antigen) with ultrasmall GO covalently modified with carnosine (GO-Car). We investigated the immunological properties of the GO-Car adjuvant in model mice. Results show that OVA@GO-Car can promote robust and durable OVA-specific antibody response, increase lymphocyte proliferation efficiency, and enhance CD4(+) T and CD8(+) T cell activation. The presence of Car in GO also probably contributes to enhancing the antigen-specific adaptive immune response through modulating the expression of some cytokines, including IL-6, CXCL1, CCL2, and CSF3. In addition, the safety of GO-Car as an adjuvant was evaluated comprehensively. No symptoms such as allergic response, inflammatory redness swelling, raised surface temperatures, physiological anomalies of blood, and remarkable weight changes were observed. Besides, after modification with carnosine, histological damages caused by GO-Car in lung, muscle, kidney, and spleen became weaken significantly. This study sufficiently suggest that GO-Car as a safe adjuvant can effectively enhance humoral and innate immune responses against antigens in vivo.

Cytosolic Delivery of Liposomal Vaccines by Means of the Concomitant Photosensitization of Phagosomes.

One of the greatest pharmaceutical challenges in vaccinology is the delivery of antigens to the cytosol of antigen-presenting cells (APCs) in order to allow for the stimulation of major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses, which may act on intracellular infections or cancer. Recently, we described a novel method for cytotoxic T-lymphocyte (CTL) vaccination by combining antigens with a photosensitizer and light for cytosolic antigen delivery. The goal of the current project was to test this immunization method with particle-based formulations. Liposomes were prepared from dipalmitoylphosphatidylcholine and cholesterol, and the antigen ovalbumin (OVA) or the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) was separately encapsulated. C57BL/6 mice were immunized intradermally with OVA liposomes or a combination of OVA and TPCS2a liposomes, and light was applied the next day for activation of the photosensitizer resulting in cytosolic release of antigen from phagosomes. Immune responses were tested both after a prime only regime and after a prime-boost scheme with a repeat immunization 2 weeks post priming. Antigen-specific CD8(+) T-cell responses and antibody responses were analyzed ex vivo by flow cytometry and ELISA methods. The physicochemical stability of liposomes upon storage and light exposure was analyzed in vitro. Immunization with both TPCS2a- and OVA-containing liposomes greatly improved CD8(+) T-cell responses as compared to immunization without TPCS2a and as measured by proliferation in vivo and cytokine secretion ex vivo. In contrast, OVA-specific antibody responses (IgG1 and IgG2c) were reduced after immunization with TPCS2a-containing liposomes. The liposomal formulation protected the photosensitizer from light-induced inactivation during storage. In conclusion, the photosensitizer TPCS2a was successfully formulated in liposomes and enabled a shift from MHC class II to MHC class I antigen processing and presentation for stimulation of strong CD8(+) T-cell responses. Therefore, photosensitive particulate vaccines may have the potential to add to current vaccine practice a new method of vaccination that, as opposed to current vaccines, can stimulate strong CD8(+) T-cell responses.

Design, Synthesis, and Evaluation of Novel 1,2,3‐Triazole‐Tethered Glycolipids as Vaccine Adjuvants

A Cu-mediated azide-alkyne click chemistry protocol was employed for the synthesis of a focused library of novel 1,2,3-triazolyl conjugates bearing various carbohydrate-steroid/triterpenoid entities. The immunogenicity of these compounds was examined initially by ex vivo assays. The lead compound 15g was further subjected to in vivo evaluation in BALB/c mice immunized with ovalbumin. These in vivo biological studies revealed an increase in B cell-mediated proliferation, higher expression levels of IL-2, TNF-α, IL-12, and IFN-γ indicating Th1 activation, together with an enhanced OVA-specific antibody (IgG) response compared to alum, affirming adjuvanticity of these glycolipids. The primary indications of response skewed toward Th1 immunity induced by the new triazoyl analogs indicate the potential of these molecules for possible application as adjuvants.

OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a, and IgG2b responses with low IgE synthesis

There is an urgent requirement for a novel vaccine that can stimulate immune responses without unwanted toxicity, including IgE elevation. We examined whether antigen ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA-NPs) with average diameter of 110nm would serve as an immune adjuvant. When BALB/c mice were immunized with OVA-NPs, they developed sufficient levels of OVA-specific IgG1 antibody responses with low levels of IgE synthesis, representing helper T (Th)2-mediated humoral immunity. OVA-specific IgG2a and IgG2b responses (i.e., Th1-mediated immunity) were also induced by secondary immunization with OVA-NPs. As expected, immunization with OVA in alum (OVA-alum) stimulated humoral immune responses, including IgG1 and IgE antibodies, with only low levels of IgG2a/IgG2b antibodies. CD4-positive T cells from mice primed with OVA-NPs produced substantial levels of IL-21 and IL-4, comparable to those from OVA-alum group. The irradiated mice receiving OVA-NPs-primed B cells together with OVA-alum-primed T cells exhibited enhanced anti-OVA IgG2b responses relative to OVA-alum-primed B cells and T cells following stimulation with OVA-NPs. Moreover, when OVA-NPs-primed, but not OVA-alum-primed, B cells were cultured in the presence of anti-CD40 monoclonal antibody, IL-4, and IL-21, or LPS plus TGF-β in vitro, OVA-specific IgG1 or IgG2b antibody responses were elicited, suggesting that immunization with OVA-NPs modulates B cells to generate IgG1 and IgG2b responses. Thus, OVA-NPs might exert their adjuvant action on B cells, and they represent a promising potential vaccine for generating both IgG1 and IgG2a/IgG2b antibody responses with low IgE synthesis.

Ultrasmall Graphene Oxide Supported Gold Nanoparticles as Adjuvants Improve Humoral and Cellular Immunity in Mice

Adjuvants play an important role in vaccines. Alum and MF59 are two dominant kinds of adjuvants used in humans. Both of them, however, have limited capacity to generate the cellular immune response required for vaccines against cancers and viral diseases. It is desirable to develop new and efficient adjuvants with the aim of improving the cellular immune response against the antigen. Here, the feasibility and efficiency of ultrasmall graphene oxide supported gold nanoparticles (usGO‐Au) as a new immune adjuvant to improve immune responses are explored. usGO‐Au is obtained from reduction of chloroauric acid using usGO and then decorated with ovalbumin (OVA, a model antigen) through physical adsorption to construct usGO‐Au@OVA. As the results show, the as‐synthesized usGO‐Au@OVA can efficiently stimulate RAW264.7 cells to secrete tumor necrosis factor‐α (TNF‐α), a mediator for cellular immune response. In vivo studies demonstrate that usGO‐Au@OVA can also promote robust OVA specific antibody response, CD8+ T cells proliferation, and different cytokines secretion. The results indicate that using usGO‐Au as an adjuvant can stimulate potent humoral and cellular immune responses against antigens, which may promote better understanding of cellular immune response and facilitate potential applications for cancer and viral vaccines.