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Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus.

ABSTRACT: In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.

SUBMITTER: Lu Z

PROVIDER: S-EPMC5629197 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus.

Lu Zhengchun Z Tallmadge Rebecca L RL Callaway Heather M HM Felippe M Julia B MJB Parker John S L JSL

Scientific reports 20171005 1

In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available i ...[more]

PMID: 28983085

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Project description:Purporse: FPV can cause a viral disease that occurs in cats, causing severe leukopenia, gastro-enteritis and nervous signs and leads to significant losses. LncRNAs biochemically resemble mRNAs posited by Jacob and Monod, yet do not template protein synthesis. Rather, lncRNAs functioning as RNA genes to play an important role in many biological and pathological processes such as inflammation and viral infection. However, lncRNAs have not been comprehensively identified in felines. Here, microarrays of mRNAs and lncRNAs were globally performed to identify the lncRNAs related to immune response of feline panleukopenia virus infection in F81 cells. Methods:lncRNA and mRNA sequencing analysis was performed on an Illumina Hiseq 4000 (LC Sciences, USA) following the vendor's recommended protocol Results: Through RNA sequencing, we performed a comprehensive analysis of lncRNAs and mRNAs in FPV infected F81 cells. F81 cells infected or uninfected FPV were used to prepare six libraries to generate 14.66 to 15.45 gigabytes (GB) clean reads per sample. We obtained 90588 novel lncRNAs, and 291 are differential expressed lncRNAs between FPV infected cells and uninfected cells. Consistent with previous studies, lncRNAs showed features distinct from mRNAs, such as shorter lengths, lesser exon numbers, and lower expression levels compared to mRNAs. The GO enrichment and KEGG pathway analyses showed that a majority of genes and lncRNAs were mainly related to the immune response of host cells with FPV infection. By differentially expressed target genes as well as co-expression network construction analysis, 21 key differentially expressed genes (19 upregulated & 2 downregulated) related to immune regulation of FPV infection in F81 cells were identified. Moreover, fifteen differentially expressed genes (five lncRNAs, ten mRNAs) were selected and independently validated by reverse-transcription qPCR and the results were consistent with the sequencing results. Conclusion: The present study is the first to provide insights into the biological connection of lncRNAs and FPV infection, which can be further explore for the potential link between lncRNA expression and the pathogenesis of FPV infection.

Project description:The feline panleukopenia virus (FPV) is a highly contagious virus that affects cats worldwide, characterized by leukopenia, high temperature and diarrhea. Recently, the continuous prevalence and variation of FPV have attracted widespread concern. The aim of this study was to investigate the isolation, genetic evolution, molecular characterization and epidemiological analysis of FPV strains among cats and dogs in China from 2019 to 2024. The 41 FPV strains, including 38 feline strains and 3 canine strains, were isolated from rectal swab samples by inoculating monolayer FK81 cells and performing a plaque purification assay. The viral and hemagglutination titers of these 41 FPV strains were 104.33~106.33 TCID50/0.1 mL and 7.0 log2~9.7 log2, respectively. Based on the complete VP2 gene, the nucleotide homology of these FPV strains was 98.91~100%, and the homology with 24 reference FPV strains from different countries and hosts was 98.85~100%. The phylogenetic analysis revealed that 41 FPV strains were more closely related to the FPV strains of Asian origin (Asian FPV strain group) than those of European and American origin (European and American FPV strain group). Furthermore, 12 mutation sites of the VP2 protein were found in these FPV strains, of which 91 and 232 amino acid sites were previously reported. Moreover, the 91 amino acid site was found to be a positive selection site with the highest dN/dS value in the selection pressure analysis. Importantly, 35 FPV strains with 91S substitution in the VP2 protein (FPV-VP2-91S strains) had formed obvious evolutionary branches in the Asian FPV strain group. The analysis of all available VP2 protein sequences of Chinese FPV strains in the GenBank database showed that the occurrence rate of FPV-VP2-91S strains had been increasing from 15.63% to 100% during 2017~2024, indicating that the FPV-VP2-91S substitution in the VP2 protein was a noteworthy molecular characteristic of the dominant FPV strains in China. These results contribute to a better understanding of their genetic evolution and renew the knowledge of FPV molecular epidemiology.

Dataset Information

Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus.

Publications

Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus.

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