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Proteomic analysis of canine vaccines

ABSTRACT: To use proteomic analysis to qualitatively and quantitatively identify mammalian protein components of commercial veterinary vaccines against canine distemper, leptospirosis, borreliosis, and rabies. A total of 25 licensed veterinary vaccines (from 4 different manufacturers) against canine distemper and leptospirosis, borreliosis, and rabies (3 year and 1 year duration of immunity) were analyzed. Duplicate samples from a single lot vial of each vaccine were prepared by acetone precipitation and proteolysis with trypsin and lysC protease mix. Peptides mixtures (one microgram) were analyzed by LC-MS using Orbitrap Fusion Lumos mass spectrometer. LC-MS data were searched against a Bos taurus protein database using MaxQuant to identify and quantify mammalian proteins in the vaccines. Protein classification and network analysis was performed of iIdentified proteins were classified by function and network analysis to visualize interactions. The largest number of mammalian proteins was identified in 3-year rabies vaccines (median 243, range 184-339) and 1 year rabies vaccines (median 193, range 169-350). Borrelia and leptospirosis-distemper (L and D) vaccines had the lowest number of proteins. Rabies vaccines had the largest number of identified proteins in common (316), 33 were unique to 1 year and 44 in 3 year products. Borrelia and L and D vaccines had 16 and 22 uniquely identified proteins, respectively. The protein classifications were primarily as modulators of protein-binding activity, enzymes, transfer/carrier proteins, cytoskeletal proteins, defense or immunity proteins, calcium-binding proteins, and extracellular matrix proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Bos Taurus (ncbitaxon:9913)

SUBMITTER: George Moore

PROVIDER: MSV000090844 | MassIVE | Tue Dec 06 08:03:00 GMT 2022

REPOSITORIES: MassIVE

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Project description:Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming M-bM-^@M-^Xwet benchM-bM-^@M-^Y techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to the C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants. Antibody profiling was performed on sera from Borrelia burgdorferi infected and non-infected humans and Peromyscus leucopus rodents against 23 variants of the surface protein OspC . For infected human serum samples, the OspC type of the infecting B. burgdorferi strain is unknown; for experimentally-infected P. leucopus serum samples, it is known. Of human serum samples, 55 were from infected individuals and 25 from naive controls. Of P. leucopus serum samples, 23 were from infected individuals and 7 were from naive controls.

Project description:Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high throughput multiplexed antibody detection from individual samples. The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal-Wallis H test for trend: p-value <0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA (median (range) Spearman’s R of 0.84 (0.65-0.95)). KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next-generation of malaria vaccines.

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Proteomic analysis of canine vaccines

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