RESUMEN
Increasing efforts are focusing on natural killer (NK) cell immunotherapies for AML. Here, we characterized CC-96191, a novel CD33/CD16a/NKG2D immune-modulating TriNKET®. CC-96191 simultaneously binds CD33, NKG2D, and CD16a, with NKG2D and CD16a co-engagement increasing the avidity for, and activation of, NK cells. CC-96191 was broadly active against human leukemia cells in a strictly CD33-dependent manner, with maximal efficacy requiring the co-engagement of CD16a and NKG2D. A frequent CD33 single nucleotide polymorphism, R69G, reduced CC-96191 potency but not maximal activity, likely because of reduced CD33 binding. Similarly, the potency, but not the maximal activity, of CC-96191 was reduced by high concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact activity. In the presence of CD33+ AML cells, CC-96191 activated NK cells but not T cells; while maximum anti-AML efficacy was similar, soluble cytokine levels were 10- to >100-fold lower than with a CD33/CD3 bispecific antibody. While CC-96191-mediated cytolysis was not affected by ABC transporter proteins, it was reduced by anti-apoptotic BCL-2 family proteins. Finally, in patient marrow specimens, CC-96191 eliminated AML cells but not normal monocytes, suggesting selectivity of TriNKET-induced cytotoxicity toward neoplastic cells. Together, these findings support the clinical exploration of CC-96191 as in NCT04789655.
RESUMEN
With the recent exception of coronavirus disease 2019 (COVID-19), tuberculosis (TB) causes more deaths globally than any other infectious disease, and approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis (Mtb). However, encouraging progress in TB vaccine development has been reported, with approximately 50% efficacy achieved in Phase 2b clinical testing of an adjuvanted subunit TB vaccine candidate. Nevertheless, current lead vaccine candidates require cold-chain transportation and storage. In addition to temperature stress, vaccines may be subject to several other stresses during storage and transport, including mechanical, photochemical, and oxidative stresses. Optimal formulations should enable vaccine configurations with enhanced stability and decreased sensitivity to physical and chemical stresses, thus reducing reliance on the cold chain and facilitating easier worldwide distribution. In this report, we describe the physicochemical stability performance of three lead thermostable formulations of the ID93 + GLA-SE TB vaccine candidate under various stress conditions. Moreover, we evaluate the impact of thermal stress on the protective efficacy of the vaccine formulations. We find that formulation composition impacts stressed stability performance, and our comprehensive evaluation enables selection of a lead single-vial lyophilized candidate containing the excipient trehalose and Tris buffer for advanced development.
RESUMEN
Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.
RESUMEN
Immunogenic agents known as adjuvants play a critical role in many vaccine formulations. Adjuvants often signal through Toll-like receptor (TLR) pathways, including formulations in licensed vaccines that target TLR4. While TLR4 is predominantly known for responding to lipopolysaccharide (LPS), a component of Gram-negative bacterial membranes, it has been shown to be a receptor for a number of molecular structures, including phospholipids. Therefore, phospholipid-based pharmaceutical formulations might have off-target effects by signaling through TLR4, confounding interpretation of pharmaceutical bioactivity. In this study we examined the individual components of a clinical stage oil-in-water vaccine adjuvant emulsion (referred to as a stable emulsion or SE) and their ability to signal through murine and human TLR4s. We found that the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) activated TLR4 and elicited many of the same immune phenotypes as canonical TLR4 agonists. This pathway was dependent on the saturation, size, and headgroup of the phospholipid. Interestingly, DMPC effects on human cells were evident but overall appeared less impactful than emulsion oil composition. Considering the prevalence of DMPC and other phospholipids used across the pharmaceutical space, these findings may contextualize off-target innate immune responses that could impact preclinical and clinical development.
RESUMEN
Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.
Asunto(s)
Antígenos de Protozoos/inmunología , Entamoeba histolytica/inmunología , Entamebiasis/inmunología , Entamebiasis/prevención & control , Vacunas Antiprotozoos/inmunología , Adyuvantes Inmunológicos/química , Administración Intranasal , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Fenómenos Químicos , Citocinas/metabolismo , Composición de Medicamentos , Entamebiasis/parasitología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunogenicidad Vacunal , Inmunoglobulina G/inmunología , Liposomas , Ratones , Vacunas Antiprotozoos/administración & dosificación , Vacunas Antiprotozoos/química , VacunaciónRESUMEN
An estimated 10 million people developed tuberculosis (TB) disease in 2019 which underscores the need for a vaccine that prevents disease and reduces transmission. The aim of our current studies is to characterize and test a prophylactic tuberculosis vaccine comprised of ID93, a polyprotein fusion antigen, and a liposomal formulation [including a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant, GLA) and QS-21] in a preclinical mouse model of TB disease. Comparisons of the ID93+GLA-LSQ vaccines are also made to the highly characterized ID93+GLA-SE oil-in-water emulsion adjuvant, which are also included these studies. The recent success of vaccine candidate M72 combined with adjuvant AS01E (GlaxoSmithKline Biologicals) in reducing progression to active disease is promising and has renewed excitement for experimental vaccines currently in the TB vaccine pipeline. The AS01E adjuvant contains monophosphoryl lipid A (MPL) and QS-21 (a saponin) in a liposomal formulation. While AS01E has demonstrated potent adjuvant activity as a component of both approved and experimental vaccines, developing alternatives to this adjuvant system will become important to fill the high demand envisioned for future vaccine needs. Furthermore, replacement sources of potent adjuvants will help to supply the demand of a TB vaccine [almost one-quarter of the world's population are estimated to have latent Mycobacterium tuberculosis (Mtb) according to the WHO 2019 global TB report], addressing (a) cost of goods, (b) supply of goods, and (c) improved efficacy of subunit vaccines against Mtb. We show that both ID93+GLA-SE (containing an emulsion adjuvant) and ID93+GLA-LSQ (containing a liposomal adjuvant) induce ID93-specific TH1 cellular immunity including CD4+CD44+ T cells expressing IFNγ, TNF, and IL-2 (using flow cytometry and intracellular cytokine staining) and vaccine-specific IgG2 antibody responses (using an ELISA). In addition, both ID93+GLA-SE and ID93+GLA-LSQ effectively decrease the bacterial load within the lungs of mice infected with Mtb. Formulations based on this liposomal adjuvant formulation may provide an alternative to AS01 adjuvant systems.
Asunto(s)
Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/uso terapéutico , Tuberculosis Pulmonar/prevención & control , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antígenos Bacterianos/uso terapéutico , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Liposomas , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Enterotoxigenic E. coli (ETEC) is a leading cause of moderate-to-severe diarrhoea. ETEC colonizes the intestine through fimbrial tip adhesin colonization factors and produces heat-stable and/or heat-labile (LT) toxins, stimulating fluid and electrolyte release leading to watery diarrhoea. We reported that a vaccine containing recombinant colonization factor antigen (CfaEB) targeting fimbrial tip adhesin of the colonization factor antigen I (CFA/I) and an attenuated LT toxoid (dmLT) elicited mucosal and systemic immune responses against both targets. Additionally, the toll-like receptor 4 ligand second-generation lipid adjuvant (TLR4-SLA) induced a potent mucosal response, dependent on adjuvant formulation. However, a combination of vaccine components at their respective individual optimal doses may not achieve the optimal immune profile. We studied a subunit ETEC vaccine prototype in mice using a response surface design of experiments (DoE), consisting of 64 vaccine dose-combinations of CfaEB, dmLT and SLA in four formulations (aqueous, aluminium oxyhydroxide, squalene-in-water stable nanoemulsion [SE] or liposomes containing the saponin Quillaja saponaria-21 [LSQ]). Nine readouts focusing on antibody functionality and plasma cell response were selected to profile the immune response of parenterally administered ETEC vaccine prototype. The data were integrated in a model to identify the optimal dosage of each vaccine component and best formulation. Compared to maximal doses used in mouse models (10 µg CfaEB, 1 µg dmLT and 5 µg SLA), a reduction in the vaccine components up to 37%, 60% and 88% for CfaEB, dmLT and SLA, respectively, maintained or even maximized immune responses, with SE and LSQ the best formulations. The DoE approach can help determine the best vaccine composition with a limited number of experiments and may accelerate development of multi-antigen/component ETEC vaccines.
RESUMEN
The growing shift to subunit antigen vaccines underscores the need for adjuvants that can enhance the magnitude and quality of immune response. Aluminum salts or alums are the first adjuvants with a long history of clinical use. Alum predominantly induces T helper 2 (TH2) type immunity in animal models, characterized by antibody production with little to no induction of antigen-specific T cells. The lack of cell-mediated or T helper 1 (TH1) immunity makes alum adjuvants ineffective in mounting durable responses against diseases like tuberculosis, malaria and HIV. Here we show that the clinically approved adjuvant, Alhydrogel, reformulated as a stable nanoparticle (nanoalum) with the anionic polymer polyacrylic acid (PAA) induces structure-dependent TH1 response against the recombinant tuberculosis antigen ID93. We found that PAA adsorption to Alhydrogel was a key parameter affecting nanoalum adjuvanticity. Adsorption depended on various factors, most notably formulation pH, and directly correlated with immunological response in mice, enhancing known hallmarks of a murine TH1 type response: induction of antigen-specific IFN-γ secreting CD4+ T cells and IgG2c subclass of antibodies. Our results demonstrate a correlation between a measurable nanoalum property and immunological response, providing a structural basis to derive a beneficial immunological outcome from a clinically approved adjuvant.
Asunto(s)
Resinas Acrílicas/química , Linfocitos T CD4-Positivos/citología , Diferenciación Celular/efectos de los fármacos , Nanopartículas/química , Células TH1/citología , Adsorción , Compuestos de Aluminio/química , Hidróxido de Aluminio/química , Óxido de Aluminio/química , Animales , Citocinas/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Inmunoglobulina G/química , Ratones , Ratones Endogámicos C57BL , Tamaño de la Partícula , Fosfatos/química , Polímeros/química , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
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.
RESUMEN
Aluminum salts, developed almost a century ago, remain the most commonly used adjuvant for licensed human vaccines. Compared to more recently developed vaccine adjuvants, aluminum adjuvants such as Alhydrogel are heterogeneous in nature, consisting of 1-10 micrometer-sized aggregates of nanoparticle aluminum oxyhydroxide fibers. To determine whether the particle size and aggregated state of aluminum oxyhydroxide affects its adjuvant activity, we developed a scalable, top-down process to produce stable nanoparticles (nanoalum) from the clinical adjuvant Alhydrogel by including poly(acrylic acid) (PAA) polymer as a stabilizing agent. Surprisingly, the PAA:nanoalum adjuvant elicited a robust TH1 immune response characterized by antigen-specific CD4+ T cells expressing IFN-γ and TNF, as well as high IgG2 titers, whereas the parent Alhydrogel and PAA elicited modest TH2 immunity characterized by IgG1 antibodies. ASC, NLRP3 and the IL-18R were all essential for TH1 induction, indicating an essential role of the inflammasome in this adjuvant's activity. Compared to microparticle Alhydrogel this nanoalum adjuvant provided superior immunogenicity and increased protective efficacy against lethal influenza challenge. Therefore PAA:nanoalum represents a new class of alum adjuvant that preferentially enhances TH1 immunity to vaccine antigens. This adjuvant may be widely beneficial to vaccines for which TH1 immunity is important, including tuberculosis, pertussis, and malaria.
RESUMEN
Influenza A annually infects 5-10% of the world's human population resulting in one million deaths. Influenza causes annual epidemics and reinfects previously exposed individuals because of antigenic drift in the glycoprotein hemagglutinin. Due to antigenic drift, the immune system is simultaneously exposed to novel and conserved parts of the influenza virus via vaccination and/or infection throughout life. Preexisting immunity has long been known to augment subsequent hemagglutination inhibitory antibody (hAb) responses. However, the preexisting immunological contributors that influence hAb responses are not understood. Therefore, we adapted and developed sequential infection and immunization mouse models using drifted influenza strains to show that MHC Class II haplotype and T-cell reactivity influences subsequent hAb responses. We found that CB6F1 mice infected with A/CA followed by immunization with A/PR8 have increased hAb responses to A/PR8 compared to C57BL/6 mice. Increased hAb responses in CB6F1 mice were CD4+ T-cell and B-cell dependent and corresponded to increased germinal center A/PR8-specific B and T-follicular helper cells. These results suggest conserved MHC Class II restricted epitopes within HA are essential for B cells to respond to drifting influenza and could be leveraged to boost hAb responses.
Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunización , Memoria Inmunológica , Virus de la Influenza A/inmunología , Animales , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Femenino , RatonesRESUMEN
BACKGROUND: Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). MATERIALS AND METHODS: In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. RESULTS: We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. CONCLUSION: These efforts will aid in the development of a platform formulation for use with other similar vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Emulsiones/química , Nanopartículas/química , Temperatura , Vacunas contra la Tuberculosis/inmunología , Animales , Formación de Anticuerpos , Química Farmacéutica , Dispersión Dinámica de Luz , Excipientes , Femenino , Liofilización , Concentración de Iones de Hidrógeno , Inmunidad Celular , Lípidos/química , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Nefelometría y Turbidimetría , Tamaño de la Partícula , Tuberculosis/inmunología , Tuberculosis/patologíaRESUMEN
Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through TLRs on B cells promotes many aspects of GC B cell responses, including affinity maturation, class switching, and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here, we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and noncanonical autophagy. Using B cell-specific αv-KO mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic hypermutation, class switching, and generation of long-lived plasma cells after immunization with virus-like particles (VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells that can be targeted to enhance antibody responses to vaccination.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Integrina alfaV/inmunología , Animales , Autofagia/inmunología , Femenino , Centro Germinal/citología , Inmunización , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/sangre , Memoria Inmunológica , Virus de la Influenza A/inmunología , Integrina alfaV/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/inmunología , Transducción de Señal/inmunología , Hipermutación Somática de Inmunoglobulina , Receptores Toll-Like/inmunología , Vacunas de Partículas Similares a Virus/inmunologíaRESUMEN
Amebiasis caused by Entamoeba histolytica is the third leading cause of parasitic mortality globally, with some 100,000 deaths annually, primarily among young children. Protective immunity to amebiasis is associated with fecal IgA and IFN-γ in humans; however, no vaccine exists. We have previously identified recombinant LecA as a potential protective vaccine antigen. Here we describe the development of a stable, manufacturable PEGylated liposomal adjuvant formulation containing two synthetic Toll-like receptor (TLR) ligands: GLA (TLR4) and 3M-052 (TLR7/8). The liposomes stimulated production of monocyte/macrophage chemoattractants MCP-1 and Mip-1ß, and Th1-associated cytokines IL-12p70 and IFN-γ from human whole blood dependent on TLR ligand composition and dose. The liposomes also demonstrated acceptable physicochemical compatibility with the recombinant LecA antigen. Whereas mice immunized with LecA and GLA-liposomes demonstrated enhanced antigen-specific fecal IgA titers, mice immunized with LecA and 3M-052-liposomes showed a stronger Th1 immune profile. Liposomes containing GLA and 3M-052 together elicited both LecA-specific fecal IgA and Th1 immune responses. Furthermore, the quality of the immune response could be modulated with modifications to the liposomal formulation based on PEG length. Compared to subcutaneous administration, the optimized liposome adjuvant composition with LecA antigen administered intranasally resulted in significantly enhanced fecal IgA, serum IgG2a, as well as systemic IFN-γ and IL-17A levels in mice. The optimized intranasal regimen provided greater than 80% protection from disease as measured by parasite antigen in the colon. This work demonstrates the physicochemical and immunological characterization of an optimized mucosal adjuvant system containing a combination of TLR ligands with complementary activities and illustrates the importance of adjuvant composition and route of delivery to enhance a multifaceted and protective immune response to amebiasis.
RESUMEN
The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain-containing adapter inducing IFN-ß are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1ß are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC-/- animals. Importantly, the early proliferation of Ag-specific CD4+ T cells was completely ablated after immunization in ASC-/- animals. Moreover, numbers of Ag-specific CD4+ T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC-/-, NLRP3-/-, and IL-1R-/- mice compared with wild-type mice and were completely ablated in TLR4-/- animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4+ T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos/inmunología , Linfocitos B/inmunología , Inflamasomas/inmunología , Células TH1/inmunología , Receptor Toll-Like 4/inmunología , Vacunas/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Femenino , Glucósidos/inmunología , Inmunidad Humoral , Interferón beta/inmunología , Interferón gamma/inmunología , Interleucina-1beta/metabolismo , Interleucina-2/inmunología , Lípido A/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Receptores Tipo I de Interleucina-1/genética , Escualeno/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/inmunología , VacunaciónRESUMEN
It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb). This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE)), in combination with rifampicin, isoniazid, and pyrazinamide (RHZ) drug treatment. We assessed the host inflammatory immune responses and lung pathology 7â»22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated with periods of enhanced bacterial control over that of drug treatment alone. Here we advance the immunotherapy model and investigate reliable correlates of protection and Mtb control.
RESUMEN
Quantification of cytokine production by CD4 and CD8 T cells after in vitro recall stimulation with the immunizing antigen is a powerful approach to characterize the cellular immune responses to immunization. Here we describe three complementary methods for such quantification including flow cytometric analysis of cytokine production by intracellular staining, ELISpot determination of the numbers of cytokine-producing cells, and generation of secreted cytokines and chemokines in culture supernatants for analysis by ELISA and/or cytometric bead arrays.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Ensayo de Immunospot Ligado a Enzimas/métodos , Inmunidad Celular , Coloración y Etiquetado/métodos , Animales , Antígenos/farmacología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Medios de Cultivo Condicionados/química , RatonesRESUMEN
Adjuvants are combined with vaccine antigens to enhance and modify immune responses, and have historically been primarily crude, undefined entities. Introducing toll-like receptor (TLR) ligands has led to a new generation of adjuvants, with TLR4 ligands being the most extensively used in human vaccines. The TLR4 crystal structures demonstrate extensive contact with their ligands and provide clues as to how they discriminate a broad array of molecules and activate or attenuate innate, as well as adaptive, responses resulting from these interactions. Leveraging this discerning ability, we made subtle chemical alterations to the structure of a synthetic monophosphoryl lipid-A molecule to produce SLA, a designer TLR4 ligand that had a number of desirable adjuvant effects. The SLA molecule stimulated human TLR4 and induced Th1 biasing cytokines and chemokines. On human cells, the activity of SLA plateaued at lower concentrations than the lipid A comparator, and induced cytokine profiles distinct from other known TLR4 agonists, indicating the potential for superior adjuvant performance. SLA was formulated in an oil-in-water emulsion, producing an adjuvant that elicited potent Th1-biased adaptive responses. This was verified using a recombinant Leishmania vaccine antigen, first in mice, then in a clinical study in which the antigen-specific Th1-biased responses observed in mice were recapitulated in humans. These results demonstrated that using structure-based approaches one can predictably design and produce modern adjuvant formulations for safe and effective human vaccines.
RESUMEN
For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.