RESUMEN
In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.
Asunto(s)
Galactosa Oxidasa , Vacunas Neumococicas , Polisacáridos Bacterianos , Streptococcus pneumoniae , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Galactosa Oxidasa/química , Galactosa Oxidasa/inmunología , Galactosa Oxidasa/metabolismo , Glicoconjugados , Ratones , Vacunas Neumococicas/química , Vacunas Neumococicas/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Serogrupo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/inmunología , Vacunas ConjugadasRESUMEN
BACKGROUND: Around 20 years ago, a 60- to 70-kDa protein was reported as a major allergen of mugwort (Artemisia vulgaris) pollen. This study was to identify and characterize its molecular properties. METHODS: Sera from 113 Chinese and 20 Dutch Artemisia-allergic/sensitized subjects (and pools thereof) were used to identify the 60- to 70-kDa allergen. Pollen extracts of seven Artemisia species were compared by immunoblotting. Transcriptomics and proteomics (mass spectrometry) of A. annua pollen were used to identify the putative 60- to 70-kDa Artemisia allergen. Both the natural purified and recombinant allergens were evaluated for IgE reactivity by ImmunoCAP. Fourteen Chinese Artemisia-allergic patients were tested intradermally with purified natural allergen. RESULTS: Immunoblots revealed two major bands at 12 and 25 kDa, and a weak band at 70 kDa for all seven Artemisia species. Using a combined transcriptomic and proteomic approach, the high molecular mass allergen in A. annua pollen was shown to be a 62-kDa putative galactose oxidase, with a putative N-glycosylation site. More than 94% of Artemisia pollen-allergic patients had IgE response to this allergen. Although recognition of a nonglycosylated recombinant version was only confirmed in a minority (16%) and at much lower IgE levels, this discrepancy cannot be explained simply by reactivity to the carbohydrate moiety on the natural allergen. Intradermal testing with the natural allergen was positive in five of nine sensitized patients. CONCLUSIONS: The previously reported 60- to 70-kDa allergen of Artemisia pollen is most likely a 62-kDa putative galactose oxidase here designated Art an 7.
Asunto(s)
Alérgenos/aislamiento & purificación , Artemisia/enzimología , Galactosa Oxidasa/inmunología , Galactosa Oxidasa/aislamiento & purificación , Adolescente , Adulto , Alérgenos/química , Alérgenos/inmunología , Artemisia/inmunología , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Polen/enzimología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Adulto JovenRESUMEN
Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3' nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.
Asunto(s)
Epítopos/genética , Epítopos/inmunología , Expresión Génica/genética , Proteínas Recombinantes de Fusión/inmunología , Transgenes/genética , Zea mays/genética , Acetiltransferasas/genética , Acetiltransferasas/inmunología , Acetiltransferasas/metabolismo , Animales , Avidina/genética , Avidina/inmunología , Avidina/metabolismo , Biolística , Western Blotting , Pollos , Hongos , Galactosa Oxidasa/genética , Galactosa Oxidasa/inmunología , Galactosa Oxidasa/metabolismo , Genes Reporteros/genética , Vectores Genéticos/genética , Inmunoensayo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptomyces , Transformación Genética , Zea mays/citología , Zea mays/metabolismoRESUMEN
Potent immunological adjuvants are urgently required to complement subunit protein and peptide vaccines in prophylactic and therapeutic vaccination. Carbonyl-amino condensations, which are essential to the inductive interaction between antigen-presenting cells and T-helper cells, were tested as a target for the enhancement or immune responses to vaccine antigens. Enzymic oxidation of cell surface galactose by the novel adjuvant NAGO, to increase amine-reactive carbonyl groups on lymphocytes and antigen-presenting cells, provided a potent noninflammatory method of enhancing the immunogenicity of viral, bacterial, and protozoal subunit vaccines in mice. In pilot protection studies with a glutathione-S-transferase vaccine against bovine Fasciola hepatica, a formulation containing NAGO as sole adjuvant agent proved very effective in inducing protection. In terms of protection in individual animals, NAGO alone was better than Quil A emulsified in squalene Montanide (five of eight animals were protected better than 80% by NAGO; four of eight animals were protected better than 93% by NAGO; two of eight animals were protected better than 80% by QA/SM). QA/SM (69% mean protection) was, however, more consistent overall than NAGO (65% mean protection). NAGO proved more effective in murine models when combined with muramyl dipeptide, but this combination has yet to be tested in protection studies.