RESUMO
Smoking remains one of the major causes of morbidity and mortality worldwide. One approach to assisting smoking cessation is via anti-nicotine vaccines, composed of nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). We have previously shown that the carrier, hapten, linker, hapten load, degree of conjugate aggregation, and presence of adducts can each influence the function (nicotine-binding capacity) of the antibody (Ab) induced. Herein, we extend those findings and show that tertiary structure is also critical to the induction of functional immune responses and that this can be influenced by conjugation conditions. We evaluated immunogenicity in mice using six lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7), and a single point (glycine 52 to glutamic acid) mutant nontoxic form of diphtheria toxin, cross-reactive material 197 (CRM197), which were synthesized under different reaction conditions resulting in conjugates with equivalent molecular characteristics (hapten load, aggregates, adducts), but a different tertiary structure. When tested in mice, better functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with conjugates with a more closed structure than those with an open conformation. These studies highlight the need for a better understanding of the physicochemical properties of small molecule conjugate vaccines.
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The anti-human immunoglobulin E (IgE) monoclonal antibody, omalizumab (Xolair®, Genentech, South San Fransisco, CA), is effective in the treatment of poorly controlled moderate to severe allergic asthma and chronic idiopathic urticaria. It acts by specifically binding to the constant domain (Cϵ3) of free human IgE in the blood and interstitial fluid. Although efficacious, use of omalizumab is limited due to restrictions on patient weight and pre-existing IgE levels, and frequent dosing (q2-4 weeks). A vaccine inducing anti-IgE antibodies has the potential for similar clinical benefits with less frequent dosing and relatively lower cost of goods. We developed a vaccine containing two IgE peptide-conjugates targeting the Cϵ3 domain of human IgE. As part of preclinical evaluation of the vaccine to optimize formulation and dose prior to initiating clinical studies, we evaluated the vaccine in non-human primates, and demonstrate the induction of anti-peptide antibodies that can bind to conformationally intact human IgE and are capable, at least in some animals, of substantial lowering circulating IgE levels.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Humanizados , Vacinas Conjugadas/imunologia , Animais , Anticorpos Monoclonais , Asma/terapia , Humanos , Omalizumab , Primatas , Urticária/terapiaRESUMO
Endosomal Toll-like receptors (TLR) such as TLR3, 7, 8 and 9 recognize pathogen associated nucleic acids. While DNA sequence does influence degree of binding to and activation of TLR9, it also appears to influence the ability of the ligand to reach the intracellular endosomal compartment. The KLK (KLKL5KLK) antimicrobial peptide, which is immunostimulatory itself, can translocate into cells without cell membrane permeabilization and thus can be used for endosomal delivery of TLR agonists, as has been shown with the IC31 formulation that contains an oligodeoxynucleotide (ODN) TLR9 agonist. We evaluated the adjuvant activity of KLK combined with CpG or non-CpG (GpC) ODN synthesized with nuclease resistant phosphorothioate (S) or native phosphodiester (O) backbones with ovalbumin (OVA) antigen in mice. As single adjuvants, CpG(S) gave the strongest enhancement of OVA-specific immunity and the addition of KLK provided no benefit and was actually detrimental for some readouts. In contrast, KLK enhanced the adjuvant effects of CpG(O) and to a lesser extent of GpC (S), which on their own had little or no activity. Indeed while CD8 T cells, IFN-γ secretion and humoral response to vaccine antigen were enhanced when CpG(O) was combined with KLK, only IFN-γ secretion was enhanced when GpC (S) was combined to KLK. The synergistic adjuvant effects with KLK/ODN combinations were TLR9-mediated since they did not occur in TLR9 knock-out mice. We hypothesize that a nuclease resistant ODN with CpG motifs has its own mechanism for entering cells to reach the endosome. For ODN without CpG motifs, KLK appears to provide an alternate mechanism for accessing the endosome, where it can activate TLR9, albeit with lower potency than a CpG ODN. For nuclease sensitive (O) backbone ODN, KLK may also provide protection from nucleases in the tissues.
RESUMO
Adjuvants are a key component in enhancing immunogenicity of vaccines and play a vital role in facilitating the induction of the correct type of immunity required for each vaccine to be optimally efficacious. Several different adjuvants are found in licensed vaccines, and many others are in pre-clinical or clinical testing. Agonists for TLRs are potent activators of the innate immune system and some, such as CpG (TLR9 agonist), are particularly good for promoting cellular immunity because of the induction of Th1 cytokines. Emulsions that have both delivery and adjuvant properties are classified as water-in-oil (W/O) or oil-in-water (O/W) formulations. The W/O emulsion Montanide ISA-51, often combined with CpG, has been widely tested in cancer vaccine clinical trials. Squalene-based O/W emulsions are in licensed influenza vaccines, and T-cell responses have been assessed pre-clinically. No clinical study has compared the two types of emulsions, and the continued use of W/O with CpG in cancer vaccines may be because the lack of single adjuvant controls has masked the interference issue. These findings may have important implications for the development of vaccines where T-cell immunity is considered essential, such as those for cancer and chronic infections. Using particulate (hepatitis B surface antigen) and soluble protein (ovalbumin) antigen, we show in mice that a W/O emulsion (ISA-51) abrogates CpG-mediated augmentation of CD8(+) T-cell responses, whereas a squalene-based O/W emulsion significantly enhanced them.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas contra Influenza/farmacologia , Manitol/análogos & derivados , Ácidos Oleicos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Animais , Feminino , Manitol/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
CONTEXT: Certain antigens, such as haptens (small molecules), short peptides, and carbohydrates (e.g. bacterial polysaccharides) are non- or poorly immunogenic unless conjugated to a carrier molecule that provides a structural scaffold for antigen presentation as well as T cell help required for B-cell activation and maturation. However, the carriers themselves are immunogenic and resulting carrier-specific immune responses may impact the immunogenicity of other conjugate vaccines using the same carrier that are administered subsequently. OBJECTIVE: Herein, using two different carriers (cross-reactive material 197, CRM and Qb-VLP), we examined in mice the impact that preexisting anti-carrier antibodies (Ab) had on subsequent immune responses to conjugates with either the same or a different carrier. METHOD: For this purpose, we used two nicotine hapten conjugates (NIC7-CRM or NIC-Qb), two IgE peptide conjugates (Y-CRM or Y-Qb), and a pneumococcal polysaccharide conjugate (Prevnar 13(®)). RESULTS: Prior exposure to CRM or Qb-VLP significantly reduced subsequent responses to the conjugated antigen having the homologous carrier, with the exception of Prevnar 13® where anti-polysaccharide responses were similar to those in animals without preexisting anti-carrier Ab. CONCLUSION: Collectively, the data suggest that the relative sizes of the antigen and carrier, as well as the conjugation density for a given conjugate impact the extent of anti-carrier suppression. All animals developed anti-carrier responses with repeat vaccination and the differences in Ab titer between groups with and without preexisting anti-carrier responses became less apparent; however, anti-carrier effects were more durable for Ab function.
Assuntos
Proteínas de Bactérias/imunologia , Haptenos/imunologia , Nicotina/imunologia , Animais , Proteínas de Bactérias/química , Feminino , Haptenos/química , Camundongos , Camundongos Endogâmicos BALB C , Nicotina/químicaRESUMO
The United States Food and Drug Administration recently removed the requirement for a General Safety Test (GST) for biologics in the Code of Federal Regulations (21 CFR 610.11). The GST, as well as abnormal toxicity (European Pharmacopeia) and innocuity tests (World Health Organization), were designed to test for extraneous toxic contaminants on each product lot intended for human use. Tests require one-week observations for general health and weight following injection of specified volumes of product batches into guinea pigs and mice. At the volumes specified, dose-related toxicity may result when the product is pharmacologically active in rodents. With vaccines, required doses may be > 3 logs higher than intended human dose on a weight-adjusted basis and if an immune modulatory adjuvant is included, systemic immune hyperactivation may cause toxicity. Herein, using the CpG/alum adjuvant combination we evaluated the different test protocols and showed their unsuitability for this adjuvant combination.
Assuntos
Produtos Biológicos/normas , Qualidade de Produtos para o Consumidor/legislação & jurisprudência , Qualidade de Produtos para o Consumidor/normas , Aprovação de Drogas/legislação & jurisprudência , Licenciamento/legislação & jurisprudência , Animais , Cobaias , Humanos , Camundongos , Testes de Toxicidade/métodos , Estados Unidos , United States Food and Drug Administration , Organização Mundial da SaúdeRESUMO
Qb bacteriophage virus-like particles (Qb-VLP) are utilized as carriers to enhance immune responses to weakly or non-immunogenic antigens such as peptides and haptens. Qb-VLPs are formed through the self-assembly of multiple Qb capsid protein monomers, a process which traps a large amount of bacterial RNA in the core of the VLP. Bacterial RNA is known to activate the innate immune system via TLR 7 and 8 found within the endosomes of certain immune cells and has been shown to contribute to the immunogenicity of Qb-VLP vaccines. Herein, we evaluated an anti-IgE vaccine comprised of two IgE peptides (Y and P) conjugated to Qb-VLP (Qb-Y and Qb-P, respectively) for in vitro stimulation of human PBMCs and in vivo immunogenicity in mice. The in vitro secretion of IFN-α from human PBMCs exposed to Qb-Y is consistent with TLR7 activation. Immunization of mice with the IgE peptide Qb-VLP conjugates induced high titers of anti-IgE antibodies in wild-type mice, but significantly lower titers in TLR7 knockout mice, supporting the self-adjuvanting role of the RNA. Inclusion of alum and alum/CpG as adjuvants partially or completely compensated for the lack of TLR7 activation in TLR7-deficient mice. Our study demonstrates the key role that TLR7 plays in the immunogenicity of the IgE peptide Qb-VLP conjugate vaccine.
RESUMO
CpG oligodeoxynucleotides (ODNs) stimulate immune cells via TLR9 and are potentially useful immunomodulators for the treatment of chronic viral infections. In the present study, different classes of CpGs were tested for their capacities for innate immune activation and antiviral activities in the woodchuck model. A class P CpG ODN was found to stimulate interferon (IFN) production in woodchuck peripheral blood mononuclear cells (PBMCs) in vitro, and following subcutaneous administration in vivo, it was observed to induce IFN and MxA expression in woodchuck PBMCs. Combination treatment with CpG ODN and entecavir (ETV) led to effective suppression of the woodchuck hepatitis virus (WHV) load in the woodchucks, with early viral responses and inhibition of replication. The woodchuck hepatitis surface antigen (WHsAg) serum concentrations were strongly decreased by CpG and ETV together but not by either agent alone, indicating synergistic effects. However, viral control post-treatment was still transient, similar to that observed with ETV alone. Significantly elevated levels of serum aspartate aminotransferase (AST) but not of alanine aminotransferase (ALT) in some of the woodchucks receiving CpG ODN were noted, but these increases were resolved before the completion of treatment and were not associated with an elevated serum bilirubin level or coagulation disorders, suggesting the absence of a significant safety concern.
Assuntos
Guanina/análogos & derivados , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Oligodesoxirribonucleotídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , DNA Viral/sangue , Modelos Animais de Doenças , Quimioterapia Combinada , Guanina/farmacologia , Anticorpos Anti-Hepatite B/sangue , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Interferon-alfa/biossíntese , Interferon-alfa/sangue , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Marmota , Oligodesoxirribonucleotídeos/genética , Distribuição AleatóriaRESUMO
Anti-nicotine vaccines comprise nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). Unfortunately, those tested clinically have failed to improve overall long term quit rates. We had shown in mice that carrier, hapten, linker, hapten load (number of haptens per carrier molecule), aggregation and adducts, as well as adjuvants influence the function of antibodies (Ab) induced. Herein, we tested an optimized antigen, NIC7-CRM, comprised of 5-aminoethoxy-nicotine (NIC7) conjugated to genetically detoxified diphtheria toxin (CRM197), with hapten load of ~16, no aggregation (~100% monomer) and minimal adducts. NIC7-CRM was tested in non-human primates (NHP) and compared to NIC-VLP, which has the same hapten and carrier as the clinical-stage CYT002-NicQb but a slightly different linker and lower hapten load. With alum as sole adjuvant, NIC7-CRM was superior to NIC-VLP for Ab titer, avidity and ex vivo function (83% and 27% nicotine binding at 40ng/mL respectively), but equivalent for in vivo function after intravenous [IV] nicotine challenge (brain levels reduced ~10%). CpG adjuvant added to NIC7-CRM/alum further enhanced the Ab responses and both ex vivo function (100% bound) and in vivo function (~80% reduction in brain). Thus, both optimal antigen design and CpG adjuvant were required to achieve a highly functional vaccine. The compelling NHP data with NIC7-CRM with alum/CpG supported human testing, currently underway.
Assuntos
Anticorpos/sangue , Proteínas de Bactérias/imunologia , Nicotina/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos , Animais , Encéfalo , Feminino , Haptenos/imunologia , Imunoconjugados/química , Macaca fascicularis , Masculino , Oligonucleotídeos , Fatores de Tempo , Vacinas SintéticasRESUMO
Anti-nicotine vaccines aim to prevent nicotine entering the brain, and thus reduce or eliminate the reward that drives nicotine addiction. Those tested in humans to date have failed to improve quit rates over placebo, possibly because antibody (Ab) responses were insufficient to sequester enough nicotine in the blood in the majority of subjects. We have previously shown in mice that the carrier, hapten and linker used in the nicotine conjugate antigen each influence the function (nicotine-binding capacity) of the Ab induced. Herein we have evaluated immunogenicity in mice of 27 lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7) and a mutant nontoxic form of diphtheria toxin (CRM197), that differed in three antigen attributes, namely hapten load (number of haptens conjugated to each molecule of CRM197), degree of conjugate aggregation and presence of adducts (small molecules attached to CRM197 via a covalent bond during the conjugation process). A range of functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with the different conjugates, which were adjuvanted with aluminum hydroxide and CpG TLR9 agonist. Trends for better functional responses in mice were obtained with conjugates having a hapten load of 11 to 18, a low level of high molecular mass species (HMMS) (i.e., not aggregated) and a low level of adducts and a more limited testing in cynomolgus monkeys confirmed these results. Thus hapten load, conjugate aggregation and presence of adducts are key antigen attributes that can influence Ab function induced by NIC7-CRM.
Assuntos
Antígenos/imunologia , Proteínas de Bactérias/imunologia , Haptenos/imunologia , Imunoglobulina G/imunologia , Nicotina/imunologia , Vacinas , Animais , Afinidade de Anticorpos , Antígenos/química , Proteínas de Bactérias/química , Encéfalo/metabolismo , Feminino , Haptenos/química , Imunoglobulina G/sangue , Macaca fascicularis , Camundongos Endogâmicos BALB C , Nicotina/sangue , Nicotina/farmacocinética , Tabagismo/terapiaRESUMO
Anti-nicotine vaccines may aid smoking cessation via the induction of anti-nicotine antibodies (Ab) which reduce nicotine entering the brain, and hence the associated reward. Ab function depends on both the quantity (titer) and the quality (affinity) of the Ab. Anti-nicotine vaccines tested previously in clinical studies had poor efficacy despite high Ab titer, and this may be due to inadequate function if Ab of low affinity were induced. In this study, we designed and synthesized a series of novel nicotine-like haptens which were all linked to diphtheria toxoid (DT) as carrier, but which differed in the site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. The resulting hapten conjugates were evaluated in a mouse model, using CpG (a TLR9 agonist) and aluminum hydroxide (Al(OH)3) as adjuvants, whereby Ab titers, affinity and function were evaluated using a radiolabeled nicotine challenge model. A series of additional linkers varying in length, rigidity and polarity were used with a single hapten to generate additional DT-conjugates, which were also tested in mice. Conjugates made with different haptens resulted in various titers of anti-nicotine Ab. Several haptens gave similarly high Ab titers, but among these, Ab affinity and hence function varied considerably. Linker also influenced Ab titer, affinity and function. These results demonstrate that immune responses induced in mice by nicotine-conjugate antigens are greatly influenced by hapten design including site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. While both Ab titer and affinity contributed to function, affinity was more sensitive to antigen differences.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Haptenos/imunologia , Nicotina/imunologia , Prevenção do Hábito de Fumar , Vacinas/imunologia , Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Animais , Anticorpos/sangue , Anticorpos/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Antígenos/química , Toxoide Diftérico/química , Toxoide Diftérico/imunologia , Feminino , Haptenos/química , Humanos , Imunoconjugados/química , Camundongos , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Nicotina/química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/imunologia , Engenharia de Proteínas/métodos , Fumar/imunologia , Relação Estrutura-Atividade , Vacinas/administração & dosagem , Vacinas/químicaRESUMO
Tobacco smoking is one of the most preventable causes of morbidity and mortality, but current smoking cessation treatments have relatively poor long term efficacy. Anti-nicotine vaccines offer a novel mechanism of action whereby anti-nicotine antibodies (Ab) in circulation prevent nicotine from entering the brain, thus avoiding the reward mechanisms that underpin nicotine addiction. Since antibody responses are typically long lasting, such vaccines could potentially lead to better long-term smoking cessation outcomes. Clinical trials of anti-nicotine vaccines to date have not succeeded, although there was evidence that very high anti-nicotine Ab titers could lead to improved smoking cessation outcomes, suggesting that achieving higher titers in more subjects might result in better efficacy overall. In this study, we evaluated CpG (TLR9 agonist) and aluminum hydroxide (Al(OH)3) adjuvants with a model anti-nicotine antigen comprising trans-3'aminomethylnicotine (3'AmNic) conjugated to diphtheria toxoid (DT). Anti-nicotine Ab titers were significantly higher in both mice and non-human primates (NHP) when 3'AmNic-DT was administered with CpG/Al(OH)3 than with Al(OH)3 alone, and affinity was enhanced in mice. CpG also improved functional responses, as measured by nicotine brain levels in mice after intravenous administration of radiolabeled nicotine (30% versus 3% without CpG), or by nicotine binding capacity of NHP antisera (15-fold higher with CpG). Further improvement should focus on maximizing Ab function, which takes into account both titer and avidity, and this may require improved conjugate design in addition to adjuvants.
Assuntos
Toxoide Diftérico/imunologia , Imunoglobulina G/imunologia , Nicotina/análogos & derivados , Nicotina/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos , Hidróxido de Alumínio/imunologia , Animais , Afinidade de Anticorpos , Ilhas de CpG/imunologia , Toxoide Diftérico/química , Feminino , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Tabagismo/terapia , Vacinas/químicaRESUMO
For the induction of robust humoral and cellular immune responses, a strong rationale exists to use vaccine-adjuvant combinations possessing both immune modulatory and enhanced delivery capabilities. Herein, we evaluated the combination of 2 different adjuvants, a TLR9 agonist, composed of synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG), and ISCOMATRIX adjuvant (ISCOMATRIX), composed of saponin, phospholipid, and cholesterol, which possesses both immunostimulatory and delivery properties. While both individual adjuvants have been shown effective in numerous preclinical and clinical studies, it is likely that for optimal adjuvant activity a combined adjuvant approach will be necessary. Herein, using three different antigens, namely, hepatitis B surface antigen (HBsAg), ovalbumin (OVA), and influenza A haemagglutinin antigen (HA), we show in mice that some adjuvant effects of CpG and ISCOMATRIX are further enhanced if they are used in combination. In particular, with all three antigens, IFN-γ levels were greatly increased with the CpG/ISCOMATRIX combination. The ability of the CpG/ISCOMATRIX combination to induce antitumor responses when administered with OVA following administration to mice of a highly metastatic OVA-secreting tumor cell line (B16-OVA melanoma) was also demonstrated. Thus the CpG/ISCOMATRIX combination may prove to be a valuable tool in the development of novel or improved vaccines.
Assuntos
Colesterol/administração & dosagem , Imunidade Inata/imunologia , Interferon gama/metabolismo , Oligodesoxirribonucleotídeos/administração & dosagem , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Animais , Antígenos/imunologia , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Camundongos , Linfócitos T/efeitos dos fármacos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Vacinas/imunologiaRESUMO
T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.
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Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Leucemia Mieloide Aguda/terapia , Manitol/análogos & derivados , Mieloma Múltiplo/terapia , Ácidos Oleicos , Oligodesoxirribonucleotídeos , Peptídeos/uso terapêutico , Adolescente , Antígenos de Neoplasias/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Feminino , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Masculino , Manitol/efeitos adversos , Mucina-1/efeitos adversos , Mucina-1/química , Mucina-1/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Mieloblastina/efeitos adversos , Mieloblastina/química , Mieloblastina/imunologia , Estadiamento de Neoplasias , Neoplasia Residual/imunologia , Neoplasia Residual/patologia , Neoplasia Residual/terapia , Ácidos Oleicos/efeitos adversos , Oligodesoxirribonucleotídeos/efeitos adversos , Oligodesoxirribonucleotídeos/imunologia , Peptídeos/efeitos adversos , Peptídeos/imunologia , Projetos Piloto , Resultado do Tratamento , Proteínas WT1/efeitos adversos , Proteínas WT1/química , Proteínas WT1/imunologiaAssuntos
Adaptação Psicológica , Anemia Falciforme , Medicina nas Artes , Fotografação , Feminino , Humanos , Adulto JovemRESUMO
Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.
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Monkeypox virus/imunologia , Infecções por Poxviridae/prevenção & controle , Vacina Antivariólica/imunologia , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina G/sangue , Macaca fascicularis/imunologia , Masculino , Testes de Neutralização , Oligodesoxirribonucleotídeos/imunologia , Infecções por Poxviridae/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
BACKGROUND: Persons infected with human immunodeficiency virus (HIV) are often hyporesponsive to immunization, including pneumococcal vaccines. We hypothesized that adding CPG 7909, a toll-like receptor 9 (TLR9) agonist and vaccine adjuvant, to 7-valent pneumococcal conjugate vaccine (7vPnC) would increase its immunogenicity in HIV-infected adults. METHODS: We performed a double-blind, placebo-controlled, phase 1b/2a trial randomizing HIV-positive patients to receive double doses of 7vPnC (Prevnar) at 0 and 3 months and 1 dose of 23-valent pneumococcal polysaccharide vaccine (PPV-23; Pneumo Novum) at 9 months, with experimental patients receiving 1 mg of CPG 7909 added to each of their 3 vaccine doses; control patients had phosphate-buffered saline added instead. Immunogenicity and safety were evaluated for up to 10 months. The primary end point was the proportion of vaccine high responders at 9 months, defined as a 2-fold increase in IgG levels to > or = 1 microg/mL for at least 5 of 7 of the 7vPnC serotypes. RESULTS: Ninety-seven participants were included in the study. The proportion of vaccine high responders was higher in the experimental group (n = 48) than among controls (n = 49; 48.8% vs 25.0%; P = .02) at 9 months. Greater proportions of high responders were also observed at 3 (51.1% vs 39.6%; P = .26), 4 (77.3% vs 56.3%; P = .03), and 10 months (87.8% vs 51.1%; P < .001). Mild systemic and injection site reactions to 7vPnC were more common in the experimental group than the control group (100% vs 81.3%; P = .002). CPG 7909 did not increase non-7vPnC IgG levels after PPV-23 immunization. No adverse effects on CD4(+) cell count or organ functions occurred in either group. CONCLUSIONS: The addition of a TLR9 agonist to 7vPnC significantly enhanced the proportion of vaccine high responders. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00562939 .
Assuntos
Adjuvantes Imunológicos/farmacologia , Infecções por HIV/imunologia , Vacinas Pneumocócicas/imunologia , Receptor Toll-Like 9/agonistas , Infecções Oportunistas Relacionadas com a AIDS/prevenção & controle , Adulto , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Método Duplo-Cego , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Imunoglobulina G/sangue , Análise de Intenção de Tratamento , Masculino , Pessoa de Meia-Idade , Vacinas Pneumocócicas/efeitos adversos , Pneumonia Pneumocócica/prevenção & controle , RNA Viral , Vacinas ConjugadasRESUMO
BACKGROUND: Chronic hepatitis C virus (HCV) infection results from weak or absent T cell responses. Pegylated-interferon-alpha (IFN-alpha) and ribavirin, the standard of care for chronic HCV, have numerous immune effects but are not potent T cell activators. A potent immune activator such as TLR9 agonist CpG oligodeoxynucleotide (CpG) may complement current treatment approaches. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from HCV chronic carriers who failed previous treatment and from healthy donors were incubated in vitro with the three main CpG classes (A, B or C), recombinant IFN-alpha-2b (IntronA) and/or ribavirin. Proliferation and cytokine secretion (IFN-alpha, IL-10 and IP-10) were evaluated. RESULTS: CpG induced proliferation and cytokine secretion in patterns expected for each CpG class with similar group means for HCV and healthy donors. IntronA and ribavirin, alone or together, had no detectable effects. IntronA and C-Class CpG together induced more IFN-alpha than CpG alone in most subjects. IFN-alpha secretion was proportional to the number of plasmacytoid dendritic cells in PBMC from healthy donors but not HCV donors in whom responses were highly heterogeneous. CONCLUSION: The strong immune stimulatory effect of CpG on PBMC isolated from treatment-failed HCV patients suggests possible utility alone or in combination with current HCV antiviral treatment.
RESUMO
Animal models are essential for acquiring safety, immunogenicity and efficacy data to support the development of novel vaccines. However, extrapolating such results to designing human trials is challenging due to species-specific differences in responses to antigens, adjuvants and pathogens. As well, most early vaccine work is conducted with in-bred mouse strains which may fail to uncover issues that could arise later in out-bred populations. Unlike drugs designed to be delivered systemically, vaccines work within a somewhat localized space, so allometric dose scaling to account for body size differences is not necessarily relevant. Comparison of immune responses and correlates of protection with a given antigen show widely variable results between animals and humans, even where protective immunity against challenge with the same pathogen can be studied. For adjuvants, it is possible to compare enhancement of immunogenicity compared to a non-adjuvant control vaccine. While some novel adjuvants provide similar levels of enhancement between species, others do not. It is also important to recognize the inter-relationship between antigens and adjuvants, since one can compensate for the other, masking particular effects. Despite all the limitations, animal immunogenicity and efficacy studies form an important part of pre-clinical development for novel vaccines, but considerable prudence is required when using and extrapolating results.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Modelos Animais , Vacinação/efeitos adversos , Vacinação/métodos , Vacinas/efeitos adversos , Vacinas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Humanos , Vacinas/administração & dosagemRESUMO
UNLABELLED: CPG 10101, a synthetic oligodeoxynucleotide (ODN), is a toll-like receptor 9 (TLR9) agonist with antiviral and immunomodulatory properties that could potentially influence chronic infection with HCV. In this multicenter Phase 1b trial, 60 HCV-positive patients (50 genotype 1 HCV) were randomized and received either placebo or CPG 10101 at 0.25, 1, 4, 10, or 20 mg subcutaneously (SC) twice weekly for 4 weeks or at 0.5 or 0.75 mg/kg SC once weekly for 4 weeks. Dose-dependent cytokine induction was observed after administration of CPG 10101. At 24 hours after administering the highest dose of 0.75 mg/kg CPG 10101, interferon (IFN)-gamma-inducible protein 10 (IP-10) had a mean increase over baseline levels (+/-SD) of 15,057 (+/-9769) pg/ml (P < 0.01, compared to placebo); IFN-alpha had a 106 (+/-63.3) pg/ml increase (P < 0.01); and 2'5'-oligoadenylate synthetase (OAS) had a 163 (+/-120.6) pmol/dl increase (P < 0.01). Decreases in HCV RNA also were dose-dependent, with the greatest group geometric mean maximum reduction of 1.69 +/- 0.618 log(10) (P < 0.05) observed in the 0.75 mg/kg dose group. Decreases >/=1 log(10) were seen in 22 of 40 patients who received >/=1 mg CPG 10101, with 3 patients exceeding a 2.5-log(10) reduction. CPG 10101 was well tolerated, and adverse events were consistent with CPG 10101's mechanism of action. CONCLUSION: In this Phase 1 study, CPG 10101 was associated with dose-dependent increases in markers of immune activation and decreases in HCV RNA levels. The data support further clinical studies of CPG 10101 for treating chronic HCV infection.