RESUMO
Deamidation of the recombinant protective antigen (rPA) correlates with decreased effectiveness of the vaccine in protecting against infection by Bacillus anthracis. We present data demonstrating dramatic deamidation of amino acid positions 713 and 719 of rPA adsorbed onto aluminum hydroxide gel, an adjuvant, relative to rPA stored in solution without adjuvant. Although deamidation did not impact total levels of rPA-specific antibodies in a mouse model, it did correlate with a decrease in toxin-neutralizing antibodies. On the basis of these data, we hypothesize that interactions of rPA with aluminum hydroxide gel are destabilizing and are the direct cause of reduced vaccine efficacy.
Assuntos
Hidróxido de Alumínio/metabolismo , Vacinas contra Antraz/metabolismo , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Adsorção , Sequência de Aminoácidos , Animais , Vacinas contra Antraz/genética , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacina contra a Peste/administração & dosagem , Vacina contra a Peste/imunologia , Peste/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/imunologia , Pós , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Feminino , Liofilização , Injeções Intradérmicas , Injeções Intramusculares , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Vacinas Sintéticas , Yersinia pestis/imunologiaRESUMO
The use of an aerosolizable form of anthrax as a biological weapon is considered to be among the most serious bioterror threats. Intranasal (IN) delivery of a dry powder anthrax vaccine could provide an effective and non-invasive administration alternative to traditional intramuscular (IM) or subcutaneous (SC) injection. We evaluated a dry powder vaccine based on the recombinant Protective Antigen (rPA) of Bacillus anthracis for vaccination against anthrax via IN immunization in a rabbit model. rPA powders were formulated and administered IN using a prototype powder delivery device. We compared serum IgG and toxin neutralizing antibody (TNA) titers of rabbits immunized IN with 10 microg rPA of a powder formulation with those immunized with the same dose of liquid rPA vaccine, delivered either IN or by IM injection. In addition, each group was tested for survival after aerosol spore challenge. Our results showed that IN vaccination with rPA powders elicited serum PA-specific IgG and TNA titers that were equivalent to those raised by liquid rPA administered IN. Serum PA-specific IgG and TNA titers after IN delivery were lower than for IM injection, however, after aerosol spore challenge, rabbits immunized IN with powders displayed 100% protection versus 63% for the group immunized IN with the liquid vaccine and 86% for the group immunized by IM injection. The results suggest that an IN powder vaccine based on rPA is at least as protective as a liquid delivered by IM injection.
Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/prevenção & controle , Bacillus anthracis/imunologia , Esporos Bacterianos/imunologia , Administração Intranasal , Aerossóis , Animais , Antraz/imunologia , Antraz/mortalidade , Vacinas contra Antraz/imunologia , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/sangue , Toxinas Bacterianas/imunologia , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pós , Coelhos , Taxa de SobrevidaRESUMO
The recombinant protective antigen (rPA) of Bacillus anthracis is a promising anthrax vaccine. We compared serum immunoglobulin G levels and toxin-neutralizing antibody titers in rabbits following delivery of various doses of vaccine by microneedle-based intradermal (i.d.) delivery or intramuscular (i.m.) injection using conventional needles. Intradermal delivery required less antigen to induce levels of antibody similar to those produced via i.m. injection during the first 2 weeks following primary and booster inoculation. This dose-sparing effect was less evident at the later stages of the immune response. Rabbits immunized i.d. with 10 mug of rPA displayed 100% protection from aerosol spore challenge, while i.m. injection of the same dose provided slightly lower protection (71%). Groups immunized with lower antigen doses were partially protected (13 to 29%) regardless of the mode of administration. Overall, our results suggest rPA formulated with aluminum adjuvant and administered to the skin by a microneedle-based device is as efficacious as i.m. vaccination.
Assuntos
Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Administração Cutânea , Animais , Feminino , Imunoglobulina G/sangue , Injeções Intramusculares , Microinjeções , Agulhas , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Vacinas SintéticasRESUMO
The purpose of this research was to prepare a dry powder vaccine formulation containing whole inactivated influenza virus (WIIV) and a mucoadhesive compound suitable for nasal delivery. Powders containing WIIV and either lactose or trehalose were produced by lyophilization. A micro-ball mill was used to reduce the lyophilized cake to sizes suitable for nasal delivery. Chitosan flakes were reduced in size using a cryo-milling technique. Milled powders were sieved between 45 and 125 microm aggregate sizes and characterized for particle size and distribution, morphology, and flow properties. Powders were blended in the micro-ball mill without the ball. Lyophilization followed by milling produced irregularly shaped, polydisperse particles with a median primary particle diameter of approximately 21 microm and a yield of approximately 37% of particles in the 45 to 125 microm particle size range. Flow properties of lactose and trehalose powders after lyophilization followed by milling and sieving were similar. Cryo-milling produced a small yield of particles in the desired size range (<10%). Lyophilization followed by milling and sieving produced particles suitable for nasal delivery with different physicochemical properties as a function of processing conditions and components of the formulation. Further optimization of particle size and morphology is required for these powders to be suitable for clinical evaluation.
Assuntos
Vacinas contra Influenza/administração & dosagem , Administração Intranasal , Liofilização , Tamanho da Partícula , Pós , Trealose/administração & dosagemRESUMO
The purpose of this research was to prepare a dry powder vaccine formulation containing whole inactivated influenza virus (VIIV) and a mucoadhesive compound suitable for nasal delivery. Powders containing WIIV and either lactose or trehalose were produced by lyophilization. A micro-ball mill was used to reduce the lyophilized cake to sizes suitable for nasal delivery. Chitosan flakes were reduced in size using a cryo-milling technique. Milled powders were sieved between 45 and 125 µm aggregate sizes and characterized for particle size and distribution, morphology, and flow properties. Powders were blended in the micro-ball mill without the ball. Lyophilization followed by milling produced irregularly shaped, polydisperse particles with a median primary particle diameter of ≈21 µm and a yield of ≈37% of particles in the 45 to 125 µm particle size range. Flow properties of lactose and trehalose powders after lyophilization followed by milling and sieving were similar. Cryo-milling produced a small yield of particles in the desired size range (<10%). Lyophilization followed by milling and sieving produced particles suitable for nasal delivery with different physicochemical properties as a function of processing conditions and components of the formulation. Further optimization of particle size and morphology is required for these powders to be suitable for clinical evaluation.
RESUMO
Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6-8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7-8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization.
Assuntos
Vacinas contra Antraz/química , Administração Intranasal , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Química Farmacêutica , Composição de Medicamentos , Estabilidade de Medicamentos , Liofilização , PósRESUMO
This paper reviews the developments in noninvasive methods of drug delivery, with a focus on the delivery of vaccines via the respiratory tract. Recent results indicate that the respiratory system, and the nasal mucosa in particular, provide a valuable target site for immunisation against respiratory and mucosal pathogens. Vaccine delivery via the nasal and pulmonary routes each present distinct sets of performance requirements. Current delivery systems in development for both routes are reviewed herein. The storage and respiratory delivery of drugs and vaccines in powder form has been shown to provide improved stability and extended retention time in the respiratory mucosa. These features, in addition to the noninvasive nature of respiratory delivery, can provide benefits to public health vaccination campaigns, facilitating mass vaccination without the high cost of maintaining cold-chain storage.
Assuntos
Sistemas de Liberação de Medicamentos , Sistema Respiratório/metabolismo , Vacinas/administração & dosagem , Administração por Inalação , Administração Intranasal , Animais , Humanos , Pós , VacinaçãoRESUMO
A new anthrax vaccine under clinical investigation is based on recombinant Bacillus anthracis protective antigen (rPA). Here, we investigated microneedle-based cutaneous and nasal mucosal delivery of rPA in mice and rabbits. In mice, intradermal (id) delivery achieved up to 90% seroconversion after a single dose, compared with 20% after intramuscular (im) injection. Intranasal (inl) delivery of a liquid formulation required 3 doses to achieve responses that were comparable with those achieved via the id or im routes. In rabbits, id delivery provided complete protection against aerosol challenge with anthrax spores; in addition, novel powder formulations administered inl provided complete protection, whereas a liquid formulation provided only partial protection. These results demonstrate, for the first time, that cutaneous or nasal mucosal administration of rPA provides complete protection against inhalational anthrax in rabbits. The novel vaccine/device combinations described here have the potential to improve the efficacy of rPA and other biodefense vaccines.
Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Vacinação , Administração Cutânea , Administração Intranasal , Animais , Vacinas contra Antraz/imunologia , Anticorpos Antibacterianos/sangue , Relação Dose-Resposta Imunológica , Sistemas de Liberação de Medicamentos , Camundongos , CoelhosRESUMO
Intranasal (i.n.) vaccination represents an attractive non-invasive alternative to needle-based injection and provides superior protection at mucosal surfaces. However, new formulations are needed to improve efficacy and reduce the refrigerated storage and distribution requirements associated with standard liquid vaccines. Here, we describe a powder formulation of whole inactivated influenza virus and a novel i.n. delivery platform. The powder-formulated vaccine elicited a significant serum antibody response in rats that was at least as strong as that provided by the liquid vaccine administered i.n. or via intramuscular (i.m.) injection. Significant nasal IgA responses were also observed solely after i.n. delivery. This study demonstrates for the first time the generation of potent nasal mucosal and systemic immune responses using an i.n. delivered influenza vaccine powder and suggests an alternative approach to vaccination against influenza and other infectious diseases.