The role of TRPA1 and TRPV1 in the perception of astringency.

Astringency, commonly described as a drying, roughening, and/or puckering sensation associated with polyphenol-rich foods affects their palatability. While the compounds eliciting astringency are known, its mechanism of action is debated. This study investigated the role of transient receptor potential (TRP) channels A1 and V1 in astringency perception. If TRP A1 or V1 have a functional role in astringency perception, then desensitizing these receptors should decrease perceived astringency. Thirty-seven panelists underwent unilateral lingual desensitization of TRP A1 and V1 channels using mustard oil and capsaicin, respectively. Panelists then evaluated four astringent stimuli: epicatechin (EC), epigallocatechin gallate (EGCG), tannic acid (TA), and potassium alum (Alum), via 2-AFC and intensity ratings. When TRPA1 receptors were desensitized on one half of the tongue via mustard oil, no significant differences were observed between the treated and untreated sides for both 2-AFC and intensity ratings. Similarly, when TRPV1 receptors were desensitized on one half of the tongue via capsaicin, no significant differences were observed between the treated and untreated sides for both 2-AFC and intensity ratings. These findings challenge the notion that TRP channels play a pivotal role in astringency perception.

Structure-Activity Relationships toward the Identification of a High-Potency Selective Human Toll-like Receptor-7 Agonist.

Toll-like receptor (TLR)-7 agonists are immunostimulatory vaccine adjuvants. A systematic structure-activity relationship (SAR) study of TLR7-active 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine led to the identification of a potent hTLR7-specific p-hydroxymethyl IMDQ 23 with an EC50 value of 0.22 µM. The SAR investigation also resulted in the identification of TLR7 selective carboxamide 12 with EC50 values of 0.32 µM for hTLR7 and 18.25 µM for hTLR8. In the vaccination study, TLR7-specific compound 23 alone or combined with alum (aluminum hydroxide wet gel) showed adjuvant activity for a spike protein immunogen in mice, with enhanced anti-spike antibody production. Interestingly, the adjuvant system comprising carboxamide 12 and alum showed prominent adjuvant activity with high levels of IgG1, IgG2b, and IgG2c in immunized mice, confirming a balanced Th1/Th2 response. In the absence of any apparent toxicity, the TLR7 selective agonists in combination with alum may make a suitable vaccine adjuvant.

The orientation of CpG conjugation on aluminum oxyhydroxide nanoparticles determines the immunostimulatory effects of combination adjuvants.

In subunit vaccines, aluminum salts (Alum) are commonly used as adjuvants, but with limited cellular immune responses. To overcome this limitation, CpG oligodeoxynucleotides (ODNs) have been used in combination with Alum. However, current combined usage of Alum and CpG is limited to linear mixtures, and the underlying interaction mechanism between CpG and Alum is not well understood. Thus, we propose to chemically conjugate Alum nanoparticles and CpG (with 5' or 3' end exposed) to design combination adjuvants. Our study demonstrates that compared to the 3'-end exposure, the 5'-end exposure of CpG in combination adjuvants (Al-CpG-5') enhances the activation of bone-marrow derived dendritic cells (BMDCs) and promotes Th1 and Th2 cytokine secretion. We used the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen (HBsAg) as model antigens to demonstrate that Al-CpG-5' enhanced antigen-specific antibody production and upregulated cytotoxic T lymphocyte markers. Additionally, Al-CpG-5' allows for coordinated adaptive immune responses even at lower doses of both CpG ODNs and HBsAg antigens, and enhances lymph node transport of antigens and activation of dendritic cells, promoting Tfh cell differentiation and B cell activation. Our novel Alum-CPG strategy points the way towards broadening the use of nanoadjuvants for both prophylactic and therapeutic vaccines.

Lycium barbarum polysaccharides-loaded Particulate Alum via Pickering emulsion as an adjuvant to enhance immune responses.

Pickering emulsion has great potential as a vaccine adjuvant due to its unique advantages such as its high antigen loading efficiency, great stability, etc. Among several adjuvants on the market, aluminum adjuvant (Alum) is the most widely used at present. However, problems such as the inability to effectively induce cellular immunity and the poor effect on subunit vaccines limit the application of Alum. As an immunopotentiator, Lycium barbarum polysaccharides (LBP) have been proven to have the ability to regulate humoral and cellular immunity. To overcome the insufficiency of Alum, we explored a new adjuvant delivery system. The Lycium barbarum polysaccharides-loaded Particulate Alum via Pickering emulsion (LBPPE) was prepared by loading Alum on the squalene/water interphase following LBP was adsorbed on the Alum surface (Fig. 10). Similar to squalene, LBPPE possesses a good biosafety profile. LBPPE was spherical with uneven surface, which increased the possibility of efficient antigen adsorption on the surface and crack of LBPPE. And the result shown that the LBPPE had high antigen loading rate at approximately 90 %. In vivo experiments, LBPPE showed an excellent ability to recruit antigen-presenting cells (APCs) at the injection sites, activate dendritic cells in the lymph nodes. Then, in the evaluation of humoral immunity, LBPPE was able to effectively induce the production of IgG, IgG1, and IgG2a. Moreover, LBPPE significantly enhanced the expression and activation of T lymphocytes, and induced a strong immune memory T cells response. All the results above suggested that LBPPE is likely to provide promising insights toward a safe and efficient adjuvant platform for vaccines.

Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity.

Anti-tumour inflammatory cytokines are highly toxic when administered systemically. Here, in multiple syngeneic mouse models, we show that the intratumoural injection of recombinantly expressed cytokines bound tightly to the common vaccine adjuvant aluminium hydroxide (alum) (via ligand exchange between hydroxyls on the surface of alum and phosphoserine residues tagged to the cytokine by an alum-binding peptide) leads to weeks-long retention of the cytokines in the tumours, with minimal side effects. Specifically, a single dose of alum-tethered interleukin-12 induced substantial interferon-γ-mediated T-cell and natural-killer-cell activities in murine melanoma tumours, increased tumour antigen accumulation in draining lymph nodes and elicited robust tumour-specific T-cell priming. Moreover, intratumoural injection of alum-anchored cytokines enhanced responses to checkpoint blockade, promoting cures in distinct poorly immunogenic syngeneic tumour models and eliciting control over metastases and distant untreated lesions. Intratumoural treatment with alum-anchored cytokines represents a safer and tumour-agnostic strategy to improving local and systemic anticancer immunity.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

Immunization with recombinant protein Ag43::UpaH with alum and 1,25(OH)2D3 adjuvants significantly protects Balb/C mice against urinary tract infection caused by uropathogenic Escherichia coli.

The majority of urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC). Designing a vaccine will certainly reduce the occurrence of infection and antibiotic resistance of the isolates. Antigen 43 (Ag43) and autotransporter H (UpaH) have been associated with the virulence of UPEC. In the present study, the efficacy of different formulations of a hybrid protein composed of Ag43 and UpaH with and without alum and 1,25(OH)2D3 (Vitamin D3) adjuvants were evaluated in mice model. A significant increase in IgG and cellular responses was developed against Ag43::UpaH as compared to the control mice. The addition of alum or a mixture of alum and Vitamin D3 to the protein significantly enhanced the serum IgG responses and tended to remain in a steady state until 6 months. In addition, the mentioned formulations produced significant amounts of IgG1, IL-4, and IL-17 as compared to the fusion protein alone. In addition to the mentioned formulations, the combination of protein with Vitamin D3 also resulted in significantly higher serum IgA and IFN-γ levels as compared to the fusion protein alone. Mice immunized with fusion plus alum and formulation protein admixed with both alum and Vitamin D3 significantly reduced the bacterial load in the bladders and kidneys of mice as compared to the control. In this study, for the first time, the ability of a novel hybrid protein in combination with adjuvants alum and Vitamin D3 was evaluated against UPEC. Our results indicated that fusion Ag43::UpaH admixed with alum and Vitamin D3 could be a promising candidate against UTIs.

CD169+ Subcapsular Macrophage Role in Antigen Adjuvant Activity.

Vaccines have played a pivotal role in improving public health, however, many infectious diseases lack an effective vaccine. Controlling the spread of infectious diseases requires continuing studies to develop new and improved vaccines. Our laboratory has been investigating the immune enhancing mechanisms of Toll-like receptor (TLR) ligand-based adjuvants, including the TLR2 ligand Neisseria meningitidis outer membrane protein, PorB. Adjuvant use of PorB increases costimulatory factors on antigen presenting cells (APC), increases antigen specific antibody production, and cytokine producing T cells. We have demonstrated that macrophage expression of MyD88 (required for TLR2 signaling) is an absolute requirement for the improved antibody response induced by PorB. Here-in, we specifically investigated the role of subcapsular CD169+ marginal zone macrophages in antibody production induced by the use of TLR-ligand based adjuvants (PorB and CpG) and non-TLR-ligand adjuvants (aluminum salts). CD169 knockout mice and mice treated with low dose clodronate treated animals (which only remove marginal zone macrophages), were used to investigate the role of these macrophages in adjuvant-dependent antibody production. In both sets of mice, total antigen specific immunoglobulins (IgGs) were diminished regardless of adjuvant used. However, the greatest reduction was seen with the use of TLR ligands as adjuvants. In addition, the effect of the absence of CD169+ macrophages on adjuvant induced antigen and antigen presenting cell trafficking to the lymph nodes was examined using immunofluorescence by determining the relative extent of antigen loading on dendritic cells (DCs) and antigen deposition on follicular dendritic cells (FDC). Interestingly, only vaccine preparations containing PorB had significant decreases in antigen deposition in lymphoid follicles and germinal centers in CD169 knockout mice or mice treated with low dose clodronate as compared to wildtype controls. Mice immunized with CpG containing preparations demonstrated decreased FDC networks in the mice treated with low dose clodronate. Conversely, alum containing preparations only demonstrated significant decreases in IgG in CD169 knockout mice. These studies stress that importance of subcapsular macrophages and their unique role in adjuvant-mediated antibody production, potentially due to an effect of these adjuvants on antigen trafficking to the lymph node and deposition on follicular dendritic cells.

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus.

BACKGROUND: Zika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV. METHODOLOGY/PRINCIPLE FINDINGS: We have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge. CONCLUSIONS/SIGNIFICANCE: These studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

The continued advance of vaccine adjuvants - 'we can work it out'.

In the last decade there have been some significant advances in vaccine adjuvants, particularly in relation to their inclusion in licensed products. This was proceeded by several decades in which such advances were very scarce, or entirely absent, but several novel adjuvants have now been included in licensed products, including in the US. These advances have relied upon several key technological insights that have emerged in this time period, which have finally allowed an in depth understanding of how adjuvants work. These advances include developments in systems biology approaches which allow the hypotheses first advanced in pre-clinical studies to be critically evaluated in human studies. This review highlights these recent advances, both in relation to the adjuvants themselves, but also the technologies that have enabled their successes. Moreover, we critically appraise what will come next, both in terms of new adjuvant molecules, and the technologies needed to allow them to succeed. We confidently predict that additional adjuvants will emerge in the coming years that will reach approval in licensed products, but that the components might differ significantly from those which are currently used. Gradually, the natural products that were originally used to build adjuvants, since they were readily available at the time of initial development, will come to be replaced by synthetic or biosynthetic materials, with more appealing attributes, including more reliable and robust supply, along with reduced heterogeneity. The recent advance in vaccine adjuvants is timely, given the need to create novel vaccines to deal with the COVID-19 pandemic. Although, we must ensure that the rigorous safety evaluations that allowed the current adjuvants to advance are not 'short-changed' in the push for new vaccines to meet the global challenge as quickly as possible, we must not jeopardize what we have achieved, by pushing less established technologies too quickly, if the data does not fully support it.

Immunoprotective Activity Induced by Leptospiral Outer Membrane Proteins in Hamster Model of Acute Leptospirosis.

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 µg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

Immune responses to HIV-1 polytope vaccine candidate formulated in aqueous and alcoholic extracts of Propolis: Comparable immune responses to Alum and Freund adjuvants.

Today's, vaccination is the most cost-effective approaches for preventing infectious diseases. In this strategy, adjuvants play an important role. Propolis from honey bee can stimulate the immune system and several studies have shown the modulating effects of Propolis on the immune responses. Here, the adjuvant effects of aqueous and alcoholic extracts of Propolis were studied on the multi-epitope vaccines against HIV-1. A recombinant vaccine against HIV-1 was prepared and BALB/c mice were immunized. subcutaneously on day 0 with 100 µl of candidate vaccine (10 µg) formulated in an alcoholic extract of Propolis. The second group of mice was immunized with the vaccine (10 µg) formulated in aqueous extract of Propolis. Also, candidate vaccine was formulated in Freund's and Alum adjuvants in the third and fourth groups. Experimental mice were immunized three times with two week intervals under the same conditions and suitable control groups. After final injection, lymphocyte proliferation was measured by BrdU method, IL-4 and IFN-γ cytokines, specific total IgG antibodies, IgG1 and IgG2a isotypes were evaluated using ELISA. The results show that the aqueous and alcoholic extracts were able to enhance lymphocyte proliferation, IL-4 and IFN-γ cytokines and antibody responses with dominant IgG1 pattern and comparable to Freund's and Alum adjuvants. It seems that aqueous and alcoholic extracts of Propolis show adjuvant activity and may be useful for vaccine formulation.

Aluminum-induced generation of lipopolysaccharide (LPS) from the human gastrointestinal (GI)-tract microbiome-resident Bacteroides fragilis.

Gram-negative bacteria of the human gastrointestinal (GI) tract microbiome: (i) are capable of generating a broad-spectrum of highly neurotoxic, pro-inflammatory and potentially pathogenic molecules; and (ii) these include a highly immunogenic class of amphipathic surface glycolipids known as lipopolysaccharide (LPS). Bacteroides fragilis (B. fragilis), a commensal, Gram negative, non-motile, non-spore forming obligatory anaerobic bacillus, and one of the most abundant bacteria found in the human GI tract, produces a particularly pro-inflammatory and neurotoxic LPS (BF-LPS). BF-LPS: (i) is known to be secreted from the B. fragilis outer membrane into the external-medium; (ii) can damage biophysiological barriers via cleavage of zonula adherens cell-cell adhesion proteins, thereby disrupting both the GI-tract barrier and the blood-brain barrier (BBB); (iii) is able to transit GI-tract barriers into the systemic circulation and cross the BBB into the human CNS; and (iv) accumulates within CNS neurons in neurodegenerative disorders such as Alzheimer's disease (AD). This short communication provides evidence that the incubation of B. fragilis with aluminum sulfate [Al2(SO4)3] is a potent inducer of BF-LPS. The results suggest for the first time that the pro-inflammatory properties of aluminum may not only be propagated by aluminum itself, but by a stimulation in the production of microbiome-derived BF-LPS and other pro-inflammatory pathogenic microbial products normally secreted from human GI-tract-resident microorganisms.

Metabolic Profiling and Compound-Class Identification Reveal Alterations in Serum Triglyceride Levels in Mice Immunized with Human Vaccine Adjuvant Alum.

Alum has been widely used as an adjuvant for human vaccines; however, the impact of Alum on host metabolism remains largely unknown. Herein, we applied mass spectrometry (MS) (liquid chromatography-MS)-based metabolic and lipid profiling to monitor the effects of the Alum adjuvant on mouse serum at 6, 24, 72, and 168 h post-vaccination. We propose a new strategy termed subclass identification and annotation for metabolomics for class-wise identification of untargeted metabolomics data generated from high-resolution MS. Using this approach, we identified and validated the levels of several lipids in mouse serum that were significantly altered following Alum administration. These lipids showed a biphasic response even 168 h after vaccination. The majority of the lipids were triglycerides (TAGs), where TAGs with long-chain unsaturated fatty acids (FAs) decreased at 24 h and TAGs with short-chain FAs decreased at 168 h. To our knowledge, this is the first report on the impact of human vaccine adjuvant Alum on the host metabolome, which may provide new insights into the mechanism of action of Alum.

ALVAC-HIV B/C candidate HIV vaccine efficacy dependent on neutralization profile of challenge virus and adjuvant dose and type.

The ALVAC-HIV clade B/AE and equivalent SIV-based/gp120 + Alum vaccines successfully decreased the risk of virus acquisition in humans and macaques. Here, we tested the efficacy of HIV clade B/C ALVAC/gp120 vaccine candidates + MF59 or different doses of Aluminum hydroxide (Alum) against SHIV-Cs of varying neutralization sensitivity in macaques. Low doses of Alum induced higher mucosal V2-specific IgA that increased the risk of Tier 2 SHIV-C acquisition. High Alum dosage, in contrast, elicited serum IgG to V2 that correlated with a decreased risk of Tier 1 SHIV-C acquisition. MF59 induced negligible mucosal antibodies to V2 and an inflammatory profile with blood C-reactive Protein (CRP) levels correlating with neutralizing antibody titers. MF59 decreased the risk of Tier 1 SHIV-C acquisition. The relationship between vaccine efficacy and the neutralization profile of the challenge virus appear to be linked to the different immunological spaces created by MF59 and Alum via CXCL10 and IL-1ß, respectively.

Adjuvants Enhance the Induction of Germinal Center and Antibody Secreting Cells in Spleen and Their Persistence in Bone Marrow of Neonatal Mice.

Immaturity of the immune system contributes to poor vaccine responses in early life. Germinal center (GC) activation is limited due to poorly developed follicular dendritic cells (FDC), causing generation of few antibody-secreting cells (ASCs) with limited survival and transient antibody responses. Herein, we compared the potential of five adjuvants, namely LT-K63, mmCT, MF59, IC31, and alum to overcome limitations of the neonatal immune system and to enhance and prolong responses of neonatal mice to a pneumococcal conjugate vaccine Pnc1-TT. The adjuvants LT-K63, mmCT, MF59, and IC31 significantly enhanced GC formation and FDC maturation in neonatal mice when co-administered with Pnc1-TT. This enhanced GC induction correlated with significantly enhanced vaccine-specific ASCs by LT-K63, mmCT, and MF59 in spleen 14 days after immunization. Furthermore, mmCT, MF59, and IC31 prolonged the induction of vaccine-specific ASCs in spleen and increased their persistence in bone marrow up to 9 weeks after immunization, as previously shown for LT-K63. Accordingly, serum Abs persisted above protective levels against pneumococcal bacteremia and pneumonia. In contrast, alum only enhanced the primary induction of vaccine-specific IgG Abs, which was transient. Our comparative study demonstrated that, in contrast to alum, LT-K63, mmCT, MF59, and IC31 can overcome limitations of the neonatal immune system and enhance both induction and persistence of protective immune response when administered with Pnc1-TT. These adjuvants are promising candidates for early life vaccination.

Alum-functionalized graphene oxide nanocomplexes for effective anticancer vaccination.

Aluminum-based adjuvant (e.g., aluminum oxyhydroxide (AlO(OH), known as the commercial Alhydrogel® (Alum)) is the first adjuvant to be used in human vaccines. Although Alum shows a robust induction of antibody-mediated immunity, its weak stimulation of cell-mediated immunity makes it a questionable adjuvant for cancer immunotherapy. Herein, we described a novel formulation of Alum-based adjuvant by preparing AlO(OH)-modified graphene oxide (GO) nanosheets (GO-AlO(OH)), which, in addition to maintaining the induction of humoral immune response by AlO(OH), could further elicit the cellular immune response by GO. Similar to Alum, GO-AlO(OH) vaccine formulation could be constructed by the incorporation of antigen using a facile mixing/adsorption approach. Antigen-loaded GO-AlO(OH) nanocomplexes facilitated cellular uptake and cytosolic release of antigens and promoted DC maturation, thereby eliciting higher antigen-specific IgG titers, inducing robust CD4+ and CD8+ T lymphocyte response, and inhibiting tumor growth in vivo. Furthermore, by employing tumor cell lysate-based cancer vaccines, GO-AlO(OH) nanocomplexes led to significant inhibition of tumor growth and can be implemented as a personalized treatment strategy for cancer vaccine development. Overall, GO-AlO(OH) nanocomplexes described herein may serve as a facile and efficient approach for effective anticancer vaccination. STATEMENT OF SIGNIFICANCE: Herein, we described a novel formulation of aluminum-based adjuvant by preparing aluminum oxyhydroxide (AlO(OH)) (known as "Alum")-modified graphene oxide (GO) nanocomplexes (GO-AlO(OH)), which, in addition to maintaining the induction of humoral immune response by AlO(OH), could further elicit the cellular immune response by GO. GO-AlO(OH) nanocomplexes can be prepared easily and in large scale by a chemical precipitation method. Similar to "Alum," antigen-loaded GO-AlO(OH) vaccine formulation could be constructed by the incorporation of antigen using a facile mixing/adsorption approach. The very simple and reproductive preparation process of vaccines and the powerful ability to raise both humoral and cellular immune responses provide a novel approach for improving cancer immunotherapy efficacy.

Correlates of GLA family adjuvants' activities.

Lipopolysaccharide (LPS) is a well-defined agonist of Toll-like receptor (TLR) 4 that activates innate immune responses and influences the development of the adaptive response during infection with Gram-negative bacteria. Many years ago, Dr. Edgar Ribi separated the adjuvant activity of LPS from its toxic effects, an effort that led to the development of monophosphoryl lipid A (MPL). MPL, derived from Salmonella minnesota R595, has progressed through clinical development and is now used in various product-enabling formulations to support the generation of antigen-specific responses in several commercial and preclinical vaccines. We have generated several synthetic lipid A molecules, foremost glucopyranosyl lipid adjuvant (GLA) and second-generation lipid adjuvant (SLA), and have advanced these to clinical trial for various indications. In this review we summarize the potential and current positioning of TLR4-based adjuvant formulations in approved and emerging vaccines.

Cationic lipids as one-component vaccine adjuvants: A promising alternative to alum.

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1ß secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants.