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TLR-7/8 agonists are a well-known class of vaccine adjuvants, with a leading example now included in Covaxin, a licensed human COVID-19 vaccine. This thereby provides the opportunity to develop newer, more potent adjuvants based on structure-function studies of these classes of compounds. Imidazoquinoline-based TLR7/8 agonists are the most potent, but when used as a vaccine adjuvant side effects can arise due to diffusion from the injection site into a systemic circulation. In this work, we sought to address this issue through structural modifications in the agonists to enhance their adsorption capacity to the classic adjuvant alum. We selected a potent TLR7-selective agonist, BBIQ (EC50 = 0.85 µM), and synthesized polyphenolic derivatives to assess their TLR7 agonistic activity and adjuvant potential alone or in combination with alum. Most of the phenolic derivatives were more active than BBIQ and, except for 12b, all were TLR7 specific. Although the synthesized compounds were less active than resiquimod, the immunization data on combination with alum, specifically the IgG1, IgG2b and IgG2c responses, were superior in comparison to BBIQ as well as the reference standard resiquimod. Compound 12b was 5-fold more potent (EC50 = 0.15 µM in TLR7) than BBIQ and induced double the IgG response to SARS-CoV-2 and hepatitis antigens. Similarly, compound 12c (EC50 = 0.31 µM in TLR7) was about 3-fold more potent than BBIQ and doubled the IgG levels. Even though compound 12d exhibited low TLR7 activity (EC50 = 5.13 µM in TLR7), it demonstrated superior adjuvant results, which may be attributed to its enhanced alum adsorption capability as compared with BBIQ and resiquimod. Alum-adsorbed polyphenolic TLR7 agonists thereby represent promising combination adjuvants resulting in a balanced Th1/Th2 immune response.
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SpikoGen® is a recombinant spike protein vaccine against COVID-19 that obtained marketing authorization in the Middle East on October 6th, 2021, becoming the first adjuvanted protein-based COVID-19 vaccine of its type to achieve approval. SpikoGen® vaccine utilizes a unique adjuvant Advax-CpG55.2, which comprises delta inulin and CpG55.2 oligonucleotide, a synthetic human toll-like receptor (TLR)-9 agonist. As part of a safety assessment, developmental and reproductive toxicity (DART) studies were undertaken in mice of Advax-CpG55.2 adjuvanted formulations including SpikoGen®, a H7 hemagglutinin influenza vaccine (rH7HA), the bivalent combination of SpikoGen® and rH7HA, and a next-generation quadrivalent spike protein vaccine. In the first study, vaccines were administered intramuscularly to pregnant dams on gestation days (GD) 6.5 and 12.5, and in the second two doses were given in the pre-mating period with a further two doses during gestation. The doses used in the pregnant mice were 250-1000 times the usual human doses on a weight for weight basis. Strong serum antibody responses with neutralizing activity against the relevant virus were seen in the immunized dams and also at the time of weaning in the sera of their pups, consistent with robust maternal antibody transfer. No adverse effects of any of the vaccine formulations were observed in the immunized dams or their pups. Notably, there were no adverse effects of any of the Advax-CpG55.2 adjuvanted vaccines on female mating performance, fertility, ovarian or uterine parameters, embryo-fetal or postnatal survival, fetal growth, or neurofunctional development. No evidence of antigen interference was observed when SpikoGen® vaccine was mixed and co-administered with influenza hemagglutinin vaccine to pregnant dams. Together with the strong safety profile of SpikoGen® vaccine seen in adults and children in human trials, this DART study data supports the safety of Advax-CpG55.2 adjuvanted COVID-19 and influenza vaccine in women of childbearing potential including during pregnancy.
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COVID-19 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Vacinas contra Influenza , Animais , Feminino , Camundongos , Gravidez , Adjuvantes Imunológicos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Vacinas contra Influenza/efeitos adversos , Glicoproteína da Espícula de CoronavírusRESUMO
There is a major unmet need for strategies to improve the immunogenicity and effectiveness of pandemic influenza vaccines, particularly in poor responder populations such as neonates. Recombinant protein approaches to pandemic influenza offer advantages over more traditional inactivated virus approaches, as they are free of problems such as egg adaptation or need for high level biosecurity containment for manufacture. However, a weakness of recombinant proteins is their low immunogenicity. We asked whether the use of an inulin polysaccharide adjuvant (Advax) alone or combined with a TLR9 agonist (CpG55.2) would enhance the immunogenicity and protection of a recombinant hemagglutinin vaccine against H7N9 influenza (rH7HA), including in neonatal mice. Advax adjuvant induced predominantly IgG1 responses against H7HA, whereas Advax-CpG55.2 adjuvant also induced IgG2a, IgG2b and IgG3 responses, consistent with the TLR9 agonist component inducing a Th1 bias. Advax-CpG55.2 adjuvanted rH7HA induced high serum neutralizing antibody titers in adult mice. In newborns it similarly overcame immune hypo-responsiveness and enhanced serum anti-rH7HA IgG levels in 7-day-old BALB/C and C57BL/6 mice. Immunized adult mice were protected against a lethal H7N9 virus challenge. When formulated with Advax-CpG55.2 adjuvant, greater protection was seen with rH7HA than with inactivated H7 whole virus antigen. Advax-CpG55.2 adjuvanted rH7HA represents a promising influenza vaccine platform for further development.
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Subtipo H7N9 do Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Camundongos , Humanos , Animais Recém-Nascidos , Hemaglutininas , Receptor Toll-Like 9 , Anticorpos Antivirais , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Adjuvantes Imunológicos , Vacinas Sintéticas , Proteínas Recombinantes , Infecções por Orthomyxoviridae/prevenção & controleRESUMO
Lipopeptides including diacylated Pam2CSK4 as well as triacylated Pam3CSK4 act as ligands of toll-like receptor (TLR)-2, a promising target for the development of vaccine adjuvants. The highly investigated Pam2CSK4 and Pam3CSK4, despite their aqueous solubility have not performed well as vaccine adjuvants which may be attributable to potential denaturation of protein antigens by these cationic surfactant-like lipopeptides. In the present investigation, we synthesized (R), (S) and racemic Pam2CS(OMe) analogs and their N-acetyl derivatives without the tetralysine component to systematically investigate the effect of stereochemistry at the thio-glycerol lipopeptide core of these lipopeptide based TLR2 agonists. The resulting compounds were compared using TLR2 reporter cell-based assays and the ability of the synthesized lipopeptides to stimulate cytokine production (IL-6, IL-10 and TNF-α) by freshly collected human PBMCs and CD40 and CD86 expressions by mouse spleen cells was also investigated. Notably, few synthesized lipopeptides were found to be potent TLR2/6 agonists, inducing cytokine production and upregulating CD40 and CD86 expressions. The TLR2/6 agonistic lipopeptides were further assessed for vaccine adjuvant effects in mice. The results confirmed that the R-stereochemistry at the thio-glycerol lipopeptide core was preferred for maximal TLR2/6 activity, as reflected in Th1 immune deviation, higher antibody levels and enhanced vaccine protection against a lethal influenza challenge.
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Toll-like receptor (TLR) agonists are promising adjuvants and the combination of TLR agonists enhance immune responses by providing synergistic immune activity via triggering different signalling pathways. However, systematic cytotoxicity due to the immediate release of such immune potentiators from the site of injection hampers its clinical performance. Nanostructured lipid carriers (NLCs) offer a possibility to incorporate multiple TLR agonists with high encapsulation efficiency and slow drug release. Herein, we synthesized NLCs from didodecyldimethylammonium bromide (D12DAB) and oleic acid and used these to co-encapsulate a Pam2CS derivative (T-2, TLR2 agonist) with an imidazoquinoline derivative (T-7, TLR7 agonist) as a combination vaccine adjuvant. Hydrodynamic diameter and zeta potential of the prepared NLCs were found to be in the range of 200-500 nm and 23-27 mV, respectively. Spherical shape and size of prepared NLCs were also assessed through Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) analysis. In-vitro release studies of T-7 demonstrated sustained release and the addition of lipopeptide T-2 augmented encapsulation efficiency (from 84 to 92.9%) with a slight trigger in the release percentage. All NLC formulations were screened in TLR2/1, TLR2/6, TLR7 and TLR8 reporter cell lines and loaded NLC formulation showed high TLR2 and TLR7 agonistic activity. Adjuvant potency was evaluated through intramuscular immunization of female C57BL/6 mice with recombinant hepatitis B surface antigen and influenza hemagglutinin protein. T-2 and T-7 loaded NLCs induced good protective efficacy in mice challenged with a lethal dose of influenza virus.
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Adjuvantes de Vacinas , Receptor 7 Toll-Like , Animais , Portadores de Fármacos , Feminino , Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Receptor 2 Toll-LikeRESUMO
Vaccines are key in charting a path out of the COVID-19 pandemic. However, development of new vaccines is highly dependent on availability of analytical methods for their design and evaluation. This paper highlights the challenges presented in having to rapidly develop vaccine analytical tools during an ongoing pandemic, including the need to address progressive virus mutation and adaptation which can render initial assays unreliable or redundant. It also discusses the potential of new computational modeling techniques to model and analyze key viral proteins and their attributes to assist vaccine production and assay design. It then reviews the current range of analytical tools available for COVID-19 vaccine application, ranging from in vitro assays for immunogen characterization to assays to measure vaccine responses in vivo. Finally, it provides a future perspective for COVID-19 vaccine analytical tools and attempts to predict how the field might evolve over the next 5-10 years.
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Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Pandemias , COVID-19/epidemiologia , COVID-19/virologia , Humanos , SARS-CoV-2/isolamento & purificaçãoRESUMO
BACKGROUND: Despite newborns being at increased risk of serious influenza infection, influenza vaccines are currently not recommended for use in infants under 6 months of age. We therefore sought to evaluate the protective efficacy in mice of an M2-based influenza vaccine (CapM2e) formulated with Advax-SM adjuvant. Vaccine protection was assessed via both passive maternal immunization and direct neonatal immunization. METHODS: For maternal transfer studies, female mice were immunized 1 week before and after mating. Blood was collected from both mother and offspring during weaning and pups were challenged when they reached 3 weeks of age with lethal doses of H1N1 and homologous reassortment influenza strain H3N2 with conserved M2. For direct immunization studies, newborns were immunized at 1 and 3 weeks of age and blood was collected prior to challenge at 4 weeks of age. RESULTS: Maternal immunization with CapM2e + Advax-SM vaccine induced high maternal M2e antibody levels that were passively transferred to their offspring and provided them with protection against both H1N1 and H3N2 influenza strains when challenged at 3 weeks of age. When used for direct immunization of neonatal mice, CapM2e + Advax-SM vaccine similarly induced high serum M2e antibody levels and protected against H1N1 and H3N2 influenza challenges with protection associated with inhibition of virus replication with a significant reduction in lung virus load in immunized pups. CONCLUSION: CapM2e + Advax-SM vaccine could be useful for protecting newborns against diverse influenza A strains, with opportunities to achieve protection by passive maternal immunization or active neonatal immunization. This data supports further development of this promising M2e-based vaccine candidate.
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Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Animais , Anticorpos Antivirais , Feminino , Imunização , Vírus da Influenza A Subtipo H3N2 , Inulina/análogos & derivados , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz ViralRESUMO
Better adjuvants are needed for vaccines against seasonal influenza. TLR7 agonists are potent activators of innate immune responses and thereby may be promising adjuvants. Among the imidazoquinoline compounds, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (BBIQ) was reported to be a highly active TLR7 agonist but has remained relatively unexplored because of its commercial unavailability. Indeed, in silico molecular modeling studies predicted that BBIQ had a higher TLR7 docking score and binding free energy than imiquimod, the gold standard TLR7 agonist. To circumvent the availability issue, we developed an improved and higher yield method to synthesize BBIQ. Testing BBIQ on human and mouse TLR7 reporter cell lines confirmed it to be TLR7 specific with significantly higher potency than imiquimod. To test its adjuvant potential, BBIQ or imiquimod were admixed with recombinant influenza hemagglutinin protein and administered to mice as two intramuscular immunizations 2 weeks apart. Serum anti-influenza IgG responses assessed by ELISA 2 weeks after the second immunization confirmed that the mice that received vaccine admixed with BBIQ had significantly higher anti-influenza IgG1 and IgG2c responses than mice immunized with antigen alone or admixed with imiquimod. This confirmed BBIQ to be a TLR7-specific adjuvant able to enhance humoral immune responses.
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Vacinas contra Influenza , Influenza Humana , Adjuvantes Imunológicos , Animais , Imiquimode , Influenza Humana/prevenção & controle , Camundongos , Receptor 7 Toll-LikeRESUMO
Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved amongâ¯>â¯50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.
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Vacinas contra Influenza/uso terapêutico , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Animais , Biologia Computacional , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Humanos , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Masculino , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Introduction: Neonates are less responsive to vaccines than adults, making it harder to protect newborns against infection. Neonatal differences in antigen-presenting cell, B and T cell function, all likely contribute. A key question is whether novel adjuvants might be able to make neonatal vaccines more effective. Areas covered: This review addresses the issues of how to improve neonatal vaccines, which we have defined as vaccines given in the first 4 weeks of life in a human infant or the first week of life in a mouse. A search was performed using keywords including 'neonatal immunity', 'neonatal immunisation', 'vaccine' and 'adjuvant' of PubMed articles published between 1960 and 2018. Expert opinion: Sugar-like structures have recently been shown to prime the infant adaptive immune system to respond to vaccines, being potentially more effective than traditional adjuvants. Sugar-based compounds with beneficial adjuvant effects in neonatal vaccine models include delta inulin (Advax), curdlan, and trehalose 6,6'-dibehenate. Such compounds make interesting neonatal adjuvant candidates, either used alone or in combination with traditional innate immune adjuvants.
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Sistema Imunitário/imunologia , Imunidade Inata/imunologia , Recém-Nascido/imunologia , Vacinação , Vacinas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Glicolipídeos/administração & dosagem , Glicolipídeos/imunologia , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/crescimento & desenvolvimento , Imunidade Inata/efeitos dos fármacos , Recém-Nascido/crescimento & desenvolvimento , Inulina/administração & dosagem , Inulina/análogos & derivados , Inulina/imunologia , Vacinas/administração & dosagem , beta-Glucanas/administração & dosagem , beta-Glucanas/imunologiaRESUMO
Insoluble, nanostructured delta inulin particles enhance the immunogenicity of co-administered protein antigens and consequently are used as a vaccine adjuvant (Advax™). To better understand their immunomodulatory properties, the in vitro hydrolysis and in vivo distribution of delta inulin particles were investigated. Delta inulin particle hydrolysis under bio-relevant acidic conditions resulted in no observable change to the bulk morphology using SEM, and HPLC results showed that only 6.1% of the inulin was hydrolysed over 21days. However, 65% of the terminal glucose groups were released, showing that acid hydrolysis relatively rapidly releases surface bound chemistries. This was used to explain in vivo biodistribution results in which delta inulin particles surface-labelled with fluorescein-5-thiosemicabizide were administered to mice using intramuscular (I.M.) or subcutaneous (S.C.) routes. Comparison analysis of the fluorescence of soluble inulin in the supernatants of homogenised tissues maintained at room temperature or heated to 100°C to solubilise particulate inulin was used to distinguish between fluorescent probe on soluble inulin and probe bound to inulin within particles. Following both I.M. and S.C. injection delta inulin exhibited a depot behaviour with local injection site residence for several weeks. Over this time, as injection site inulin reduced, there was measurable transport of intact delta inulin particles by macrophages to secondary lymphoid organs and the liver. Ultimately, the injected delta inulin became solubilised resulting in its detection in the plasma and in the urine. Thus injected delta inulin particles are initially taken up by macrophages at the site of injection, trafficked to secondary lymphoid tissue and the liver, and hydrolysed resulting in their becoming soluble and diffusing into the blood stream, from whence they are glomerularly filtered and excreted into the urine. These results provide important insights into the biodistribution of I.M. or S.C. injected delta inulin particles when used as vaccine adjuvants and their method of excretion.
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Adjuvantes Imunológicos/metabolismo , Inulina/metabolismo , Inulina/urina , Adjuvantes Imunológicos/administração & dosagem , Animais , Metabolismo dos Carboidratos , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Hidrólise , Imunomodulação , Vacinas contra Influenza/administração & dosagem , Injeções Intramusculares , Inulina/administração & dosagem , Inulina/química , Tecido Linfoide/metabolismo , Camundongos , Monócitos/metabolismo , Infecções por Orthomyxoviridae/prevenção & controle , Absorção Subcutânea , Distribuição TecidualRESUMO
The serious consequences of influenza infection during pregnancy have been recognized for almost a century. In this article, we reviewed the evidence on the immunogenicity, safety and impact of maternal influenza immunization for both mother and child. After vaccination, pregnant women have similar protective titers of anti-influenza antibodies as non-pregnant women, demonstrating that pregnancy does not alter the trivalent inactivated influenza vaccine immune response. Studies from the United States, Europe and resource-constrained regions demonstrate that maternal vaccination is associated with increased anti-influenza antibody concentrations and protection in the newborn child as well as the immunized mother. Given the acceptable safety profile of influenza vaccines and the World Health Organization's recommendation for its use in pregnant women, maternal vaccination with inactivated influenza vaccine is a cost-effective approach to decrease influenza disease in newborns. However, as seen for influenza immunization in the elderly, the protective efficacy of current inactivated vaccines in protection of newborns is 50% at best, indicating significant room for vaccine improvement, which could potentially be achieved by addition of a safe and effective adjuvant. Thus, global deployment of inactivated influenza immunization during pregnancy would have substantial and measurable health benefits for mothers and their newborns.
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Esquemas de Imunização , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Mães , Complicações Infecciosas na Gravidez/prevenção & controle , Europa (Continente) , Feminino , Humanos , Recém-Nascido , Vacinas contra Influenza/administração & dosagem , Gravidez , Resultado do Tratamento , Estados UnidosRESUMO
γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB.
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Glicolipídeos/imunologia , Ativação Linfocitária/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Tuberculose/imunologia , Imunidade Adaptativa/imunologia , Animais , Antígenos de Bactérias/imunologia , Hemiterpenos/imunologia , Metilglucosídeos/imunologia , Compostos Organofosforados/imunologia , Polissacarídeos/imunologia , Subpopulações de Linfócitos T/microbiologia , Tuberculose/microbiologiaRESUMO
Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.
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Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Ectromelia Infecciosa/transmissão , Animais , Terapia de Imunossupressão , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RecidivaRESUMO
Mucosal-associated invariant T (MAIT) cells have a semi-invariant TCR Vα-chain, and their optimal development is dependent upon commensal flora and expression of the nonpolymorphic MHC class I-like molecule MR1. MAIT cells are activated in an MR1-restricted manner by diverse strains of bacteria and yeast, suggesting a widely shared Ag. Recently, human and mouse MR1 were found to bind bacterial riboflavin metabolites (ribityllumazine [RL] Ags) capable of activating MAIT cells. In this study, we used MR1/RL tetramers to study MR1 dependency, subset heterogeneity, and protective effector functions important for tuberculosis immunity. Although tetramer(+) cells were detected in both MR1(+/+) and MR1(-/-) TCR Vα19i-transgenic (Tg) mice, MR1 expression resulted in significantly increased tetramer(+) cells coexpressing TCR Vß6/8, NK1.1, CD44, and CD69 that displayed more robust in vitro responses to IL-12 plus IL-18 and RL Ag, indicating that MR1 is necessary for the optimal development of the classic murine MAIT cell memory/effector subset. In addition, tetramer(+) MAIT cells expressing CD4, CD8, or neither developing in MR1(+/+) Vα19i-Tg mice had disparate cytokine profiles in response to RL Ag. Therefore, murine MAIT cells are considerably more heterogeneous than previously thought. Most notably, after mycobacterial pulmonary infection, heterogeneous subsets of tetramer(+) Vα19i-Tg MAIT cells expressing CXCR3 and α4ß1 were recruited into the lungs and afforded early protection. In addition, Vα19iCα(-/-)MR(+/+) mice were significantly better protected than were Vα19iCα(-/-)MR1(-/-), wild-type, and MR1(-/-) non-Tg mice. Overall, we demonstrate considerable functional diversity of MAIT cell responses, as well as that MR1-restricted MAIT cells are important for tuberculosis protective immunity.
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Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade nas Mucosas , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Riboflavina/imunologia , Tuberculose/imunologia , Tuberculose/veterinária , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos Ly/genética , Antígenos Ly/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Heterogeneidade Genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Memória Imunológica , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-18/genética , Interleucina-18/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Mycobacterium bovis/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Riboflavina/análogos & derivados , Riboflavina/farmacologia , Transdução de Sinais , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbpΔ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbpΔ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.
Assuntos
Citocinas/genética , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/imunologia , Células Th2/metabolismo , Proteínas Virais/genética , Animais , Linhagem Celular , Citocinas/metabolismo , Suscetibilidade a Doenças , Vírus da Ectromelia/genética , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/mortalidade , Ectromelia Infecciosa/virologia , Técnicas de Silenciamento de Genes , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais , Proteínas Virais/imunologiaRESUMO
Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8α(+)CD8ß(-/lo), implying predominant expression of CD8αα homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH2OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in Vα19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-ß repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.
Assuntos
Antígenos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/química , Humanos , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Proteínas Mutantes/metabolismo , Redobramento de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
Tuberculosis remains one of the top three leading causes of morbidity and mortality worldwide, complicated by the emergence of drug-resistant Mycobacterium tuberculosis strains and high rates of HIV coinfection. It is important to develop new antimycobacterial drugs and immunomodulatory therapeutics and compounds that enhance antituberculous immunity. Dipterinyl calcium pentahydrate (DCP), a calcium-complexed pterin compound, has previously been shown to inhibit human breast cancer cells and hepatitis B virus (HBV). DCP inhibitory effects were attributed to induction of apoptosis and/or increased production of interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we tested the ability of DCP to mediate inhibition of intracellular mycobacteria within human monocytes. DCP treatment of infected monocytes resulted in a significant reduction in viability of intracellular but not extracellular Mycobacterium bovis BCG. The antimicrobial activity of DCP was comparable to that of pyrazinamide (PZA), one of the first-line antituberculosis drugs currently used. DCP potentiated monocyte antimycobacterial activity by induction of the cysteine-cysteine (C-C) chemokine macrophage inflammatory protein 1ß (MIP-1ß) and inducible nitric oxide synthase 2. Addition of human anti-MIP-1ß neutralizing antibody or a specific inhibitor of the l-arginase-nitric oxide pathway (N(G)-monomethyl l-arginine [l-NMMA] monoacetate) reversed the inhibitory effects of DCP on intracellular mycobacterial growth. These findings indicate that DCP induced mycobacterial killing via MIP-1ß- and nitric oxide-dependent effects. Hence, DCP acts as an immunoregulatory compound enhancing the antimycobacterial activity of human monocytes.
Assuntos
Antibacterianos/farmacologia , Quimiocina CCL4/metabolismo , Monócitos/microbiologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Óxido Nítrico/metabolismo , Pteridinas/farmacologia , Animais , Quimiocina CCL4/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Monócitos/metabolismo , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Human γ(9)δ(2) T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ(9)δ(2) T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ(9)δ(2) T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ(9)δ(2) T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ(9)δ(2) T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ(9)δ(2) T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ(9)δ(2) T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.
Assuntos
Granzimas/metabolismo , Macrófagos/enzimologia , Monócitos/enzimologia , Mycobacterium/fisiologia , Subpopulações de Linfócitos T/enzimologia , Células Cultivadas , Regulação Bacteriana da Expressão Gênica , Técnicas de Silenciamento de Genes , Granzimas/genética , Granzimas/farmacologia , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Mycobacterium/efeitos dos fármacos , Testes de Neutralização , RNA Interferente Pequeno/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Two doses of either trivalent live attenuated or inactivated influenza vaccines (LAIV and TIV, respectively) are approved for young children (≥ 24 months old for LAIV and ≥ 6 months old for TIV) and induce protective antibody responses. However, whether combinations of LAIV and TIV are safe and equally immunogenic is unknown. Furthermore, LAIV is more protective than TIV in children for unclear reasons. METHODS: Children 6-35 months old were administered, 1 month apart, 2 doses of either TIV or LAIV, or combinations of LAIV and TIV in both prime/boost sequences. Influenza-specific antibodies were measured by hemagglutination inhibition (HAI), and T cells were studied in flow cytometric and functional assays. Highly conserved M1, M2, and NP peptides predicted to be presented by common HLA class I and II were used to stimulate interferon-γ enzyme-linked immunospot responses. RESULTS: All LAIV and/or TIV combinations were well tolerated and induced similar HAI responses. In contrast, only regimens containing LAIV induced influenza-specific CD4(+), CD8(+), and γδ T cells, including T cells specific for highly conserved influenza peptides. CONCLUSIONS: Prime/boost combinations of LAIV and TIV in young children were safe and induced similar protective antibodies. Only LAIV induced CD4(+), CD8(+), and γδ T cells relevant for broadly protective heterosubtypic immunity. CLINICAL TRIALS REGISTRATION: NCT00231907.