TLR4 Agonist Adjuvant Research Articles

A synthetic TLR4 agonist significantly increases humoral immune responses and the protective ability of an MDCK-cell-derived inactivated H7N9 vaccine in mice.

Antigenically divergent H7N9 viruses pose a potential threat to public health, with the poor immunogenicity of candidate H7N9 vaccines demonstrated in clinical trials underscoring the urgent need for more-effective H7N9 vaccines. In the present study, mice were immunized with various doses of a suspended-MDCK-cell-derived inactivated H7N9 vaccine, which was based on a low-pathogenic H7N9 virus, to assess cross-reactive immunity and cross-protection against antigenically divergent H7N9 viruses. We found that the CRX-527 adjuvant, a synthetic TLR4 agonist, significantly enhanced the humoral immune responses of the suspended-MDCK-cell-derived H7N9 vaccine, with significant antigen-sparing and immune-enhancing effects, including robust virus-specific IgG, hemagglutination-inhibiting (HI), neuraminidase-inhibiting (NI), and virus-neutralizing (VN) antibody responses, which are crucial for protection against influenza virus infection. Moreover, the CRX-527-adjuvanted H7N9 vaccine also elicited cross-protective immunity and cross-protection against a highly pathogenic H7N9 virus with a single vaccination. Notably, NI and VN antibodies might play an important role in cross-protection against lethal influenza virus infections. This study showed that a synthetic TLR4 agonist adjuvant has a potent immunopotentiating effect, which might be considered worth further development as a means of increasing vaccine effectiveness.

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals

BackgroundPlasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development.MethodsMice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay.ResultsIn the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer.ConclusionPfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

Vaccine-linked chemotherapy improves cardiac structure and function in a mouse model of chronic Chagas disease.

Chagas disease, caused by chronic infection with the protozoan parasite Trypanosoma cruzi, affects 6-7 million people worldwide. The major clinical manifestation of Chagas disease is chronic Chagasic cardiomyopathy (CCC), which encompasses a spectrum of symptoms including arrhythmias, hypertrophy, dilated cardiomyopathy, heart failure, and sudden death. Current treatment is limited to two antiparasitic drugs, benznidazole (BNZ) and nifurtimox, but both have limited efficacy to halt the progression of CCC. We developed a vaccine-linked chemotherapy strategy using our vaccine consisting of recombinant Tc24-C4 protein and a TLR-4 agonist adjuvant in a stable squalene emulsion, in combination with low dose benznidazole treatment. We previously demonstrated in acute infection models that this strategy parasite specific immune responses, and reduced parasite burdens and cardiac pathology. Here, we tested our vaccine-linked chemotherapy strategy in a mouse model of chronic T. cruzi infection to evaluate the effect on cardiac function. Female BALB/c mice infected with 500 blood form T. cruzi H1 strain trypomastigotes were treated beginning 70 days after infection with a low dose of BNZ and either low or high dose of vaccine, in both sequential and concurrent treatments streams. Control mice were untreated, or administered only one treatment. Cardiac health was monitored throughout the course of treatment by echocardiography and electrocardiograms. Approximately 8 months after infection, endpoint histopathology was performed to measure cardiac fibrosis and cellular infiltration. Vaccine-linked chemotherapy improved cardiac function as evidenced by amelioration of altered left ventricular wall thickness, left ventricular diameter, as well as ejection fraction and fractional shortening by approximately 4 months of infection, corresponding to two months after treatment was initiated. At study endpoint, vaccine-linked chemotherapy reduced cardiac cellular infiltration, and induced significantly increased antigen specific IFN-γ and IL-10 release from splenocytes, as well as a trend toward increased IL-17A. These data suggest that vaccine-linked chemotherapy ameliorates changes in cardiac structure and function induced by infection with T. cruzi. Importantly, similar to our acute model, the vaccine-linked chemotherapy strategy induced durable antigen specific immune responses, suggesting the potential for a long lasting protective effect. Future studies will evaluate additional treatments that can further improve cardiac function during chronic infection.

Heterologous mRNA-protein vaccination with Tc24 induces a robust cellular immune response against Trypanosoma cruzi, characterized by an increased level of polyfunctional CD8+ T-cells

Tc24 is a Trypanosoma cruzi-derived flagellar protein that, when formulated with a TLR-4 agonist adjuvant, induces a balanced immune response in mice, elevating IgG2a antibody titers and IFN-γ levels. Furthermore, vaccination with the recombinant Tc24 protein can reduce parasite levels and improve survival during acute infection. Although some mRNA vaccines have been proven to elicit a stronger immune response than some protein vaccines, they have not been used against T. cruzi. This work evaluates the immunogenicity of a heterologous prime/boost vaccination regimen using protein and mRNA-based Tc24 vaccines. Mice (C57BL/6) were vaccinated twice subcutaneously, three weeks apart, with either the Tc24-C4 protein + glucopyranosyl A (GLA)-squalene emulsion, Tc24 mRNA Lipid Nanoparticles, or with heterologous protein/mRNA or mRNA/protein combinations, respectively. Two weeks after the last vaccination, mice were euthanized, spleens were collected to measure antigen-specific T-cell responses, and sera were collected to evaluate IgG titers and isotypes. Heterologous presentation of the Tc24 antigen generated antigen-specific polyfunctional CD8+ T cells, a balanced Th1/Th2/Th17 cytokine profile, and a balanced humoral response with increased serum IgG, IgG1 and IgG2c antibody responses. We conclude that heterologous vaccination using Tc24 mRNA to prime and Tc24-C4 protein to boost induces a broad and robust antigen-specific immune response that was equivalent or superior to two doses of a homologous protein vaccine, the homologous mRNA vaccine and the heterologous Tc24-C4 Protein/mRNA vaccine.

E6020, a TLR4 Agonist Adjuvant, Enhances Both Antibody Titers and Isotype Switching in Response to Immunization with Hapten-Protein Antigens and Is Diminished in Mice with TLR4 Signaling Insufficiency.

The mechanisms by which TLR4-based adjuvants enhance immunogenicity are not fully understood. We have taken advantage of a novel knock-in mouse strain that homozygously expresses two single-nucleotide polymorphisms (SNPs) that are homologous to human TLR4 (rs4986790 and rs4986791) and have been associated with LPS hyporesponsiveness in vivo and in vitro. TLR4-SNP (coexpressing mutations D298G/N397I in TLR4) mice that recapitulate the human phenotype were compared with wild-type (WT) mice for their hapten-specific Ab responses after immunization with hapten 4-hydroxy-3-nitrophenyl acetyl (NP) NP-Ficoll or NP-OVA in the absence or presence of a water-soluble TLR4 analog adjuvant, E6020. IgM and IgG anti-NP responses were comparable in WT and TLR4-SNP mice after immunization with either NP-Ficoll or NP-OVA only. E6020 significantly yet transiently improved the IgM and IgG anti-NP responses of both WT and TLR4-SNP mice to NP-Ficoll (T-independent), with modestly enhanced Ab production in WT mice. In contrast, T-dependent (NP-OVA), adjuvant-enhanced responses showed sustained elevation of NP-specific Ab titers in WT mice, intermediate responses in TLR4-SNP mice, and negligible enhancement in TLR4-/- mice. E6020-enhanced early humoral responses in WT and TLR4-SNP mice to NP-OVA favored an IgG1 response. After a second immunization, however, the immune responses of TLR4-SNP mice remained IgG1 dominant, whereas WT mice reimmunized with NP-OVA and E6020 exhibited increased anti-NP IgG2c titers and a sustained increase in the IgG1 and IgG2c production by splenocytes. These findings indicate that E6020 increases and sustains Ab titers and promotes isotype class switching, as evidenced by reduced titers and IgG1-dominant immune responses in mice with TLR4 insufficiency.

Design of a Multi-Epitopes Vaccine against Hantaviruses: An Immunoinformatics and Molecular Modelling Approach.

Hantaviruses are negative-sense, enveloped, single-stranded RNA viruses of the family Hantaviridae. In recent years, rodent-borne hantaviruses have emerged as novel zoonotic viruses posing a substantial health issue and socioeconomic burden. In the current research, a reverse vaccinology approach was applied to design a multi-epitope-based vaccine against hantavirus. A set of 340 experimentally reported epitopes were retrieved from Virus Pathogen Database and Analysis Resource (ViPR) and subjected to different analyses such as antigenicity, allergenicity, solubility, IFN gamma, toxicity, and virulent checks. Finally, 10 epitopes which cleared all the filters used were linked with each other through specific GPGPG linkers to construct a multi-antigenic epitope vaccine. The designed vaccine was then joined to three different adjuvants—TLR4-agonist adjuvant, β-defensin, and 50S ribosomal protein L7/L12—using an EAAAK linker to boost up immune-stimulating responses and check the potency of vaccine with each adjuvant. The designed vaccine structures were modelled and subjected to error refinement and disulphide engineering to enhance their stability. To understand the vaccine binding affinity with immune cell receptors, molecular docking was performed between the designed vaccines and TLR4; the docked complex with a low level of global energy was then subjected to molecular dynamics simulations to validate the docking results and dynamic behaviour. The docking binding energy of vaccines with TLR4 is −29.63 kcal/mol (TLR4-agonist), −3.41 kcal/mol (β-defensin), and −11.03 kcal/mol (50S ribosomal protein L7/L12). The systems dynamics revealed all three systems to be highly stable with a root-mean-square deviation (RMSD) value within 3 Å. To test docking predictions and determine dominant interaction energies, binding free energies of vaccine(s)–TLR4 complexes were calculated. The net binding energy of the systems was as follows: TLR4-agonist vaccine with TLR4 (MM–GBSA, −1628.47 kcal/mol and MM–PBSA, −37.75 kcal/mol); 50S ribosomal protein L7/L12 vaccine with TLR4 complex (MM–GBSA, −194.62 kcal/mol and MM–PBSA, −150.67 kcal/mol); β-defensin vaccine with TLR4 complex (MM–GBSA, −9.80 kcal/mol and MM–PBSA, −42.34 kcal/mol). Finally, these findings may aid experimental vaccinologists in developing a very potent hantavirus vaccine.

TLR4 Agonist Combined with Trivalent Protein JointS of Streptococcus suis Provides Immunological Protection in Animals.

Streptococcus suis (S. suis) serotype 2 (SS2) is the causative agent of swine streptococcosis and can cause severe diseases in both pigs and humans. Although the traditional inactive vaccine can protect pigs from SS2 infection, novel vaccine candidates are needed to overcome its shortcomings. Three infection-associated proteins in S. suis—muramidase-released protein (MRP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and DLD, a novel putative dihydrolipoamide dehydrogenase—have been previously identified by immunoproteomic assays. In this study, the effective immune protection of the recombinant trivalent protein GAPDH-MRP-DLD (JointS) against SS2, SS7, and SS9 was determined in zebrafish. To improve the immune efficacy of JointS, monophosphoryl lipid A (MPLA) as a TLR4 agonist adjuvant, which induces a strong innate immune response in the immune cells of mice and pigs, was combined with JointS to immunize the mice. The results showed that immunized mice could induce the production of a high titer of anti-S. suis antibodies; as a result, 100% of mice survived after SS2 infection. Furthermore, JointS provides good protection against virulent SS2 strain infections in piglets. Given the above, there is potential to develop JointS as a novel subunit vaccine for piglets to prevent infection by SS2 and other S. suis serotypes.

The TLR4 agonist adjuvant SLA-SE promotes functional mucosal antibodies against a parenterally delivered ETEC vaccine

Many pathogens establish infection at mucosal surfaces such as the enteric pathogen Enterotoxigenic E. coli (ETEC). Thus, there is a pressing need for effective vaccination strategies that promote protective immunity at mucosal surfaces. Toll-like receptor (TLR) ligands have been extensively developed as vaccine adjuvants to promote systemic immunity, whereas attenuated bacterial toxins including cholera toxin and heat-labile toxin (LT) have initially been developed to promote mucosal immunity. Here we evaluate the ability of the TLR4 agonist second-generation lipid adjuvant formulated in a stable emulsion (SLA-SE) to augment functional mucosal antibodies elicited by intramuscular immunization with a recombinant ETEC vaccine antigen. We find that, in mice, parenterally delivered SLA-SE is at least as effective as the double-mutant LT (LTR192G/L211A, dmLT) adjuvant in promoting functional antibodies and eliciting intestinal IgA responses to the vaccine antigen. In addition, SLA-SE enhanced both the IgG2a response in the mucosa and serum, and the production of LT neutralizing serum antibodies elicited by dmLT four to eightfold. These results reveal unexpected mucosal adjuvant properties of this TLR4 agonist adjuvant when delivered intramuscularly. This may have a substantial impact on the development of vaccines against enteric and other mucosal pathogens.

The TLR-4 agonist adjuvant, GLA-SE, improves magnitude and quality of immune responses elicited by the ID93 tuberculosis vaccine: first-in-human trial

Tuberculosis (TB) is the leading cause of infectious death worldwide. Development of improved TB vaccines that boost or replace BCG is a major global health goal. ID93 + GLA-SE is a fusion protein TB vaccine candidate combined with the Toll-like Receptor 4 agonist adjuvant, GLA-SE. We conducted a phase 1, randomized, double-blind, dose-escalation clinical trial to evaluate two dose levels of the ID93 antigen, administered intramuscularly alone or in combination with two dose levels of the GLA-SE adjuvant, in 60 BCG-naive, QuantiFERON-negative, healthy adults in the US (ClinicalTrials.gov identifier: NCT01599897). When administered as 3 injections, 28 days apart, all dose levels of ID93 alone and ID93 + GLA-SE demonstrated an acceptable safety profile. All regimens elicited vaccine-specific humoral and cellular responses. Compared with ID93 alone, vaccination with ID93 + GLA-SE elicited higher titers of ID93-specific antibodies, a preferential increase in IgG1 and IgG3 subclasses, and a multifaceted Fc-mediated effector function response. The addition of GLA-SE also enhanced the magnitude and polyfunctional cytokine profile of CD4+ T cells. The data demonstrate an acceptable safety profile and indicate that the GLA-SE adjuvant drives a functional humoral and T-helper 1 type cellular response.

A lipid A-based TLR4 mimetic effectively adjuvants a Yersinia pestis rF-V1 subunit vaccine in a murine challenge model

Vaccination can significantly reduce worldwide morbidity and mortality to infectious diseases, thereby reducing the health burden as a result of microbial infections. Effective vaccines contain three components: a delivery system, an antigenic component of the pathogen, and an adjuvant. With the growing use of purely recombinant or synthetic antigens, there is a need to develop novel adjuvants that enhance the protective efficacy of a vaccine against infection. Using a structure–activity relationship (SAR) model, we describe here the synthesis of a novel TLR4 ligand adjuvant compound, BECC438, by bacterial enzymatic combinatorial chemistry (BECC). This compound was identified using an in vitro screening pipeline consisting of (i) NFκB activation and cytokine production by immortalized cell lines, (ii) cytokine production by primary human PBMCs, and (iii) upregulation of surface costimulatory markers by primary human monocyte-derived dendritic cells. Using this SAR screening regimen, BECC438 was shown to produce an innate immune activation profile comparable to the well-characterized TLR4 agonist adjuvant compound, phosphorylated hexa-acyl disaccharide (PHAD). To evaluate the in vivo adjuvant activity of BECC438, we used the known protective Yersinia pestis (Yp) antigen, rF1-V, in a murine prime-boost vaccination schedule followed by lethal challenge. In addition to providing protection from lethal challenge, BECC438 stimulated production of higher levels of rF1-V-specific total IgG as compared to PHAD after both prime and boost vaccinations. Similar to PHAD, BECC438 elicited a balanced IgG1/IgG2c response, indicative of active TH2/TH1-driven immunity. These data demonstrate that the novel BECC-derived TLR4L adjuvant, BECC438, elicits cytokine profiles in vitro similar to PHAD, induces high antigen-specific immune titers and a TH1-associated IgG2c immune titer skew, and protects mice against a lethal Yp challenge.

A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations.

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants

Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific molecules contribute to the stimulatory activity. QS-21 is a defined, highly purified, and soluble saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. We observed QS-21 to elicit caspase-1-dependent IL-1β and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, our data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1β/IL-18 release. At higher concentrations, QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixture of saponins also induced IL-1β in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, we have identified QS-21 as a nonparticulate single molecular saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigen-specific responses in vivo.

Vaccination Produces CD4 T Cells with a Novel CD154-CD40-Dependent Cytolytic Mechanism.

The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust Th1 responses to a variety of vaccine Ags and is in clinical development for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein Ag and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly, these in vivo CTLs were CD4 T cells, not CD8 T cells, and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs, the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the Fas ligand-Fas, TRAIL-DR5, and canonical death pathways, indicating a novel mechanism of CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40L) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs, which kill through a previously unknown CD154-dependent mechanism.

A first-in-human phase 1 dose-escalating trial of G305 in patients with solid tumors expressing NY-ESO-1.

Human tumor cells express antigens that serve as targets for the host cellular immune system. This phase 1 dose-escalating study was conducted to assess safety and tolerability of G305, a recombinant NY-ESO-1 protein vaccine mixed with glucopyranosyl lipid A (GLA), a synthetic TLR4 agonist adjuvant, in a stable emulsion (SE). Twelve patients with solid tumors expressing NY-ESO-1 were treated using a 3 + 3 design. The NY-ESO-1 dose was fixed at 250 µg, while GLA-SE was increased from 2 to 10 µg. Safety, immunogenicity, and clinical responses were assessed prior to, during, and at the end of therapy. G305 was safe and immunogenic at all doses. All related AEs were Grade 1 or 2, with injection site soreness as the most commonly reported event (100%). Overall, 75% of patients developed antibody response to NY-ESO-1, including six patients with increased antibody titer ( ≥ 4-fold rise) and three patients with seroconversion from negative (titer < 100) to positive (titer ≥ 100). CD4 T-cell responses were observed in 44.4% of patients; 33.3% were new responses and 1 was boosted ( ≥ 2-fold rise). Following treatment, 8 of 12 patients had stable disease for 3 months or more; at the end of 1 year, three patients had stable disease and nine patients were alive. G305 is a potent immunotherapeutic agent that can stimulate NY-ESO-1-specific antibody and T-cell responses. The vaccine was safe at all doses of GLA-SE (2–10 µg) and showed potential clinical benefit in this population of patients.

Evaluating the efficacy of rBmHATαc as a multivalent vaccine against lymphatic filariasis in experimental animals and optimizing the adjuvant formulation

Developing an effective vaccine against lymphatic filariasis will complement the WHO's effort to eradicate the infection from endemic areas. Currently 83 different countries are endemic for this infection and over 1 billion people are at risk. An effective vaccine coupled with mass drug administration will reduce the morbidity and social stigma associated with this gruesome disease. Several potential vaccine candidates that can confer partial protection in experimental animals have been reported from different laboratories. However, no licensed vaccines are currently available for this disease. Among the several vaccine antigens identified from our laboratory, three most promising antigens; rBmHSPαc (α crystalline domain and c-terminal extension of Heat Shock Protein 12.6), rBmALT-2 (Abundant larval transcript) and rBmTSP LEL (Tetraspanin large extracellular loop) was further developed as a recombinant fusion protein vaccine (rBmHATαc). In a mouse model this fusion protein vaccine gave close to 68% protection following a challenge infection. To improve the vaccine efficiency of rBmHATαc, in this study we evaluated various preparations of alum (AL007, AL019, Alhydrogel and Imject® Alum) as adjuvants. Our results show that mice immunized with rBmHATαc formulated in AL007 (alum from IDRI) and/or AL019 (alum plus TLR-4 agonist from IDRI) gave the highest IgG antibody titer compared to other groups. Subsequent in vivo challenge experiments confirmed that >95% protection can be achieved when AL007 or AL019 was used as the adjuvant. However, when Imject® Alum or alhydrogel was used as the adjuvant only 76% and 72% protection respectively could be achieved. These results show that AL007 or AL019 (IDRI) is an excellent choice of adjuvant for the rBmHATαc vaccine against B. malayi L3 in mice.

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly-functional T(H)1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR-domain-containing adapter inducing IFN-beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T(H)1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA-SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild-type mice. We find that both MyD88 and TRIF are necessary for GLA-SE to induce a poly-functional T(H)1 immune response characterized by CD4(+) T cells producing IFN-γ, TNF, and IL-2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA-SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T(H)1-skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

Microneedle mediated intradermal delivery of adjuvanted recombinant HIV-1 CN54gp140 effectively primes mucosal boost inoculations

Dissolving polymeric microneedle arrays formulated to contain recombinant CN54 HIVgp140 and the TLR4 agonist adjuvant MPLA were assessed for their ability to elicit antigen-specific immunity. Using this novel microneedle system we successfully primed antigen-specific responses that were further boosted by an intranasal mucosal inoculation to elicit significant antigen-specific immunity. This prime-boost modality generated similar serum and mucosal gp140-specific IgG levels to the adjuvanted and systemic subcutaneous inoculations. While the microneedle primed groups demonstrated a balanced Th1/Th2 profile, strong Th2 polarization was observed in the subcutaneous inoculation group, likely due to the high level of IL-5 secretion from cells in this group. Significantly, the animals that received a microneedle prime and intranasal boost regimen elicited a high level IgA response in both the serum and mucosa, which was greatly enhanced over the subcutaneous group. The splenocytes from this inoculation group secreted moderate levels of IL-5 and IL-10 as well as high amounts of IL-2, cytokines known to act in synergy to induce IgA. This work opens up the possibility for microneedle-based HIV vaccination strategies that, once fully developed, will greatly reduce risk for vaccinators and patients, with those in the developing world set to benefit most.