Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

Hansen, Debra T.; Robida, Mark D.; Craciunescu, Felicia M.; Loskutov, Andrey; Rodenberry, John Charles; Wang, Xiao; Olson, Tien L.; Patel, Hetal; Fromme, Petra; Sykes, Kathryn

doi:10.1038/srep21925

Cited by 10 publications

(39 citation statements)

References 65 publications

(89 reference statements)

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“…This method allows the transfer of a foreign gene quickly and reliably. It can also be used to produce an antibody specifically against a protein without many purification steps [24]. We previously reported that antibodies produced by genetic immunization using human nephrin DNA had high specificity [25,26].…”

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confidence: 99%

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

Takeuchi

Naito

Kawashima

et al. 2017

Nephron

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Background: Focal segmental glomerulosclerosis (FSGS) is considered a subset of the podocytopathies. The molecular pathogenesis of podocytopathy is still unknown. There has not been an experimental animal model of isolated podocytopathy induced by antibody in C57BL/6 strain, which is widely used as the genetic background. Nephrin is closely associated with the slit diaphragm of the glomerular podocyte, and has recently received attention as a potential therapeutic target. The function of nephrin, especially its role in FSGS development via podocytopathy, is being elucidated. We report our experience with a C57BL/6 FSGS model induced by polyclonal rabbit anti-mouse nephrin antibody (α-mNep Ab). Methods: α-mNep Ab, which was generated by genetic immunization, was administered into C57BL/6 mice at once, intravenously. Urinary protein excretion, the development of glomerulosclerosis and the number of podocyte in mouse kidney were evaluated. Results: The α-mNep Ab-induced FSGS was associated with massive proteinuria and nephrotic syndrome. In periodic acid-Schiff staining, FSGS was observed from day 7 after antibody injection. Podocyte numbers and podocyte marker (anti-Wilms tumor 1 and anti-synaptopodin)-positive areas were clearly decreased. These results suggest that this FSGS mouse model reliably reproduces the human nephrotic syndrome and FSGS. Conclusion: We succeeded in making the nephrotic syndrome model mice induced by α-mNep Ab using C57BL/6. This model may be useful for studying the mechanisms of podocytopathy.

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confidence: 99%

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

Takeuchi

Naito

Kawashima

et al. 2017

Nephron

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“…We recently reported the first description of the efficiency of any DNA immunization approach specific for membrane proteins (Hansen et al, 2016). We applied the DNA-gold micronanoplex method (Svarovsky et al, 2009) and the immunization vector pCMVi-LSrCOMPTT (Table 1; Figure 1) to a structurally diverse set of 17 membrane proteins from two Biosafety Level 3 pathogens, and demonstrated polyclonal antibody production for more than half of the targets (Hansen et al, 2016).…”

Section: Strategic Planningmentioning

confidence: 99%

“…We applied the DNA-gold micronanoplex method (Svarovsky et al, 2009) and the immunization vector pCMVi-LSrCOMPTT (Table 1; Figure 1) to a structurally diverse set of 17 membrane proteins from two Biosafety Level 3 pathogens, and demonstrated polyclonal antibody production for more than half of the targets (Hansen et al, 2016). A major validation of this study was that antibody production correlated with prior reports of target immunogenicity in infected or immunized hosts (Hansen et al, 2016). To support widespread use, all plasmid vectors from this study have been made available through the non-profit DNASU Plasmid Repository (Seiler et al, 2014).…”

Section: Strategic Planningmentioning

confidence: 99%

“…Our recent report (Hansen et al, 2016) included pCMVi-LS-LTA-R192G and pCMVi-LS-LTB (Table 1), which encode mouse-codon-optimized sequences of the enterotoxigenic E. coli heat-labile enterotoxin subunits A (inactive mutant R192G) and B that are fused to the α 1 -antitrypsin leader sequence (Whitlock et al, 2011). These vectors have also been applied to vaccine development (Whitlock et al, 2011); however, the effect of these vectors on antibody titers has not been specifically evaluated (Whitlock et al, 2011; Hansen et al, 2016). For soluble (i.e., non-transmembrane) proteins, inclusion of the adjuvant vectors pCMVi-mGMCSF and pCMVi-Flt3L (Table 1) led to improved titers (Chambers and Johnston, 2003).…”

Section: Strategic Planningmentioning

confidence: 99%

“…CpGs can be included with plasmid DNA during incorporation into bullets (Hansen et al, 2016). CpG oligodeoxynucleotides with complete phosphorothioate linkages, which resist nuclease digestion, can be purchased (e.g., Invitrogen or Sigma) in desalted form and without further purification.…”

Section: Strategic Planningmentioning

confidence: 99%

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Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

et al. 2018

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Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA-based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA-gold micronanoplexes, which are complexes of micron-sized gold particles that allow dermal penetration and nanometer-sized gold particles that provide a higher surface area for DNA binding than micron gold alone. In contrast to antibodies derived from immunizations with detergent-solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.

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RNA Transcription and Splicing Errors as a Source of Cancer Frameshift Neoantigens for Vaccines

Shen

Zhang

Lee

et al. 2019

Sci Rep

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the success of checkpoint inhibitors in cancer therapy is largely attributed to activating the patient's immune response to their tumor's neoantigens arising from DnA mutations. this realization has motivated the interest in personal cancer vaccines based on sequencing the patient's tumor DnA to discover neoantigens. Here we propose an additional, unrecognized source of tumor neoantigens. We show that errors in transcription of microsatellites (MS) and mis-splicing of exons create highly immunogenic frameshift (fS) neoantigens in tumors. the sequence of these fS neoantigens are predictable, allowing creation of a peptide array representing all possible neoantigen fS peptides. this array can be used to detect the antibody response in a patient to the FS peptides. A survey of 5 types of cancers reveals peptides that are personally reactive for each patient. this source of neoantigens and the method to discover them may be useful in developing cancer vaccines. Checkpoint inhibitor immunotherapeutics are revolutionizing cancer therapy. However, even in the most responsive cancers a substantial portion (50-80%) of the patients have poor to no positive response 1-5. A surprising finding in the analysis of these patients was that one of the best correlates of response has been the total number of neoantigens in the tumor 6-8. This is also the case for patients with high microsatellite instability (MSI) where the production of FS neoantigens drives the effective anti-tumor immune responses 9-11. The realization of the immunological importance of these DNA mutations has spawned the effort to develop personal vaccines 12. As promising as early studies are of these vaccines, a major problem is that the majority of tumors will not have enough neoantigen-generating mutations to sustain development of a personal vaccine 13-15. For example, melanoma tumors have a high mutational level with an average of 200 neoepitope mutations. This provides a large number to algorithmically screen for optimal antigenic presentation. In recent reports of two Phase I clinical trials of personal melanoma vaccines, starting with 90~2,000 personal neoantigens, 10 or 20 were identified for the vaccine 16,17. However, in glioblastoma multiforme (GBM) only 3.5% patients had a high tumor mutation load, and further analysis showed that only a very small subset of GBM patients would potentially benefit from checkpoint blockade treatment 18. This is also consistent with a lack of response in GBM patients to checkpoint inhibitors 19. Massive genomic sequencing results indicated that GBM, ovarian cancer, breast adenocarcinoma and many other cancer types had very low number non-synonymous mutations, which will make these cancers difficult targets for personalized cancer vaccines 14,20. To solve this problem, we have investigated an alternative source of neoantigens which could possibly expand the scope of the application and efficacy of the neoantigen based cancer vaccines. In the process of becoming a tumor, not only does the DNA mutation rate increase wit...

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Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

Cited by 10 publications

References 65 publications

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

RNA Transcription and Splicing Errors as a Source of Cancer Frameshift Neoantigens for Vaccines

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