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1.
Immun Inflamm Dis ; 4(2): 135-147, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27957325

RESUMO

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/terapia
2.
Vaccine ; 34(50): 6292-6300, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27816374

RESUMO

We evaluated 52 different E. coli expressed pneumococcal proteins as immunogens in a BALB/c mouse model of S. pneumoniae lung infection. Proteins were selected based on genetic conservation across disease-causing serotypes and bioinformatic prediction of antibody binding to the target antigen. Seven proteins induced protective responses, in terms of reduced lung burdens of the serotype 3 pneumococci. Three of the protective proteins were histidine triad protein family members (PhtB, PhtD and PhtE). Four other proteins, all bearing LPXTG linkage domains, also had activity in this model (PrtA, NanA, PavB and Eng). PrtA, NanA and Eng were also protective in a CBA/N mouse model of lethal pneumococcal infection. Despite data inferring widespread genomic conservation, flow-cytometer based antisera binding studies confirmed variable levels of antigen expression across a panel of pneumococcal serotypes. Finally, BALB/c mice were immunized and intranasally challenged with a viulent serotype 8 strain, to help understand the breadth of protection. Those mouse studies reaffirmed the effectiveness of the histidine triad protein grouping and a single LPXTG protein, PrtA.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Sequência Conservada , Testes Genéticos , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/imunologia , Animais , Antígenos de Bactérias/genética , Carga Bacteriana , Proteínas de Bactérias/genética , Clonagem Molecular , Biologia Computacional , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Expressão Gênica , Pulmão/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Pneumonia Pneumocócica/microbiologia , Streptococcus pneumoniae/genética , Análise de Sobrevida
3.
Immunopharmacol Immunotoxicol ; 38(3): 184-96, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27121368

RESUMO

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/química
4.
Vaccines (Basel) ; 4(1)2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26805897

RESUMO

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.

5.
Int Immunopharmacol ; 29(2): 663-671, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26404190

RESUMO

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 Sintéticas
6.
Int Immunopharmacol ; 25(2): 518-27, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25737198

RESUMO

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/terapia
7.
PLoS One ; 8(10): e76557, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098532

RESUMO

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/química
8.
J Infect Dis ; 208(2): 319-29, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23596321

RESUMO

BACKGROUND: We evaluated the immunological responses of African green monkeys immunized with multiple F and G protein-based vaccines and assessed protection against the Memphis 37 strain of respiratory syncytial virus (RSV). METHODS: Monkeys were immunized with F and G proteins adjuvanted with immunostimulatory (CpG) oligodeoxyribonucleotides admixed with either Alhydrogel or ISCOMATRIX adjuvant. Delivery of F and G proteins via replication incompetent recombinant vesicular stomatitis viruses (VSVs) and human adenoviruses was also evaluated. Mucosally or parenterally administered recombinant adenoviruses were used in prime-boost regimens with adjuvanted proteins or recombinant DNA. RESULTS: Animals primed by intranasal delivery of recombinant adenoviruses, and boosted by intramuscular injection of adjuvanted F and G proteins, developed neutralizing antibodies and F/G protein-specific T cells and were protected from RSV infection. Intramuscular injections of Alhydrogel (plus CpG) adjuvanted F and G proteins reduced peak viral loads in the lungs of challenged monkeys. Granulocyte numbers were not significantly elevated, relative to controls, in postchallenge bronchoalveolar lavage samples from vaccinated animals. CONCLUSIONS: This study has validated the use of RSV (Memphis 37) in an African green monkey model of intranasal infection and identified nonreplicating vaccines capable of eliciting protection in this higher species challenge model.


Assuntos
Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/farmacologia , Vírus Sinciciais Respiratórios/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/imunologia , Lavagem Broncoalveolar/métodos , Chlorocebus aethiops , Granulócitos/imunologia , Granulócitos/virologia , Imunização/métodos , Pulmão/imunologia , Pulmão/virologia , Distribuição Aleatória , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/genética , Linfócitos T/imunologia , Linfócitos T/virologia , Vesiculovirus/genética , Vesiculovirus/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Carga Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
9.
Int Immunopharmacol ; 16(1): 50-6, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23562759

RESUMO

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/química
10.
J Immunol ; 189(3): 1467-79, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22745376

RESUMO

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.


Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Elementos de DNA Transponíveis/imunologia , Retrovirus Endógenos/imunologia , Vacinas contra a AIDS/genética , Adulto , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/genética , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
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