RESUMO
Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.
Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Influenza Humana/prevenção & controle , Neuraminidase/genética , Vacinas Sintéticas/genética , RNARESUMO
PURPOSE: Nasal delivery is a favorable route for vaccination against most respiratory infections, as antigen deposited in the nasal turbinate and Waldeyer's ring areas induce mucosal and systemic immune responses. However, little is known about the nasal distribution of the vaccines, specifically for infants. METHODS: Anatomical nasal replicas of five subjects, 3-24 months, were developed to assess local intranasal vaccine delivery using MAD Nasal™ device, and understand impact of breathing conditions and administration parameters. High performance liquid chromatography was used to quantify the deposition pattern and determine the delivery efficiency. RESULTS: The delivery efficiency on average for all models was found to be 86.57±14.23%. There were no significant differences in the total delivery efficiency between the models in all cases. However, the regional deposition pattern was altered based on the model and subsequent administration. Furthermore, removing the foam tip from the MAD Nasal™ device, to study the impact of insertion length, did not significantly increase the efficiency within the two models tested, 5- and 16-month. CONCLUSION: Incorporating nasal replicas in testing provided a benchmark to determine the efficiency of a common intranasal vaccine delivery combination product. This proposed platform would allow comparing other potential nasal vaccine delivery devices.
Assuntos
Modelos Anatômicos , Mucosa Nasal/metabolismo , Vacinação/métodos , Vacinas/farmacocinética , Administração Intranasal , Pré-Escolar , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Lactente , Nariz/anatomia & histologia , Nariz/diagnóstico por imagem , Impressão Tridimensional , Tomografia Computadorizada por Raios X , Vacinas/administração & dosagemRESUMO
Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280â¯nm and their emission spectra collected from 290 to 400â¯nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4⯵g/mL and limit of linearity between 100 and 200⯵g/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.
Assuntos
Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Bordetella pertussis/química , Medições Luminescentes/métodos , Adesinas Bacterianas/análise , Adesinas Bacterianas/química , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/química , Fímbrias Bacterianas/química , Fluorescência , Humanos , Triptofano/química , Tirosina/química , Vacinas/imunologia , Fatores de Virulência de Bordetella/análise , Fatores de Virulência de Bordetella/químicaRESUMO
The pneumococcal histidine triad protein D (PhtD) is believed to play a central role in pneumococcal metal ion homeostasis and has been proposed as a promising vaccine candidate against pneumococcal disease. To investigate for potential stabilizers, a panel of physiologically relevant metals was screened using the thermal shift assay and it was found that only Zn2+ and Mn2+ were able to increase PhtD melting temperature. Differential scanning calorimetry analysis revealed a sequential unfolding of PhtD and the presence of at least 3 independent folding domains that can be stabilized by Zn2+ and Mn2+. UV spectroscopy and fluorescence quenching studies showed significant Zn2+-induced tertiary structure changes in PhtD characterized by decreased accessibility of inner tryptophan residues to the aqueous solvent. Isothermal titration calorimetry data show no apparent binding to Mn2+ but revealed a Zn2+:PhtD exothermic interaction stoichiometry of 3:1 with strong enthalpic contribution, suggesting that 3 of the 5 histidine triads are accessible binding sites for Zn2+. Only Zn+2, but not Mn+2, was able to increase the thermal stability of PhtD in the presence of aluminum hydroxide adjuvant, making it a promising stabilizer excipient candidate in vaccine products containing PhtD.
Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Manganês/química , Streptococcus pneumoniae/metabolismo , Zinco/química , Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Anticorpos Antibacterianos/química , Proteínas de Transporte/química , Histidina/química , Vacinas Pneumocócicas/química , Triptofano/químicaRESUMO
Monitoring the immunological functionality of vaccine formulations is critical for vaccine development. While the traditional approach using established animal models has been relatively effective, the use of animals is costly and cumbersome, and animal models are not always reflective of a human response. The development of a human-based approach would be a major step forward in understanding how vaccine formulations might behave in humans. Here, we describe a platform methodology using fresh human whole blood (hWB) to monitor adjuvant-modulated, antigen-specific responses to vaccine formulations, which is amenable to analysis by standard immunoassays as well as a variety of other analytical techniques.