Production of Novel Monoclonal Antibodies Against the SERINC5 HIV-1 Restriction Factor

Summary

In this article, Molnar et al. developed a series of novel monoclonal antibodies against the multi-pass transmembrane protein SERINC5. Later, they demonstrated that these SERINC5 mAbs can be used to detect endogenously expressed SERINC5 protein in various cell lines and furtherly showed some of the antibodies could detect SERINC5 that is presented in HIV-1 viral stocks. Monoclonal antibodies obtained in this work could provide tools to study the functions and mechanisms of SERINC5 and potentially be engineered to serve as therapeutic tools.

Background

The serine incorporator (SERINC) protein family has five members that localize to cell membranes. Previous studies have shown that SERINC3 and more potently, SERINC5, can effectively restrict HIV-1 infection. However, the detection of endogenous SERINC proteins in cells has been limited so far as to the lack of suitable antibodies, thus posed a challenge for the study of the proteins. To solve this problem, Molnar et al. utilized a DNA-prime/peptide boost immunization regimen in mice to produce monoclonal antibodies (mAbs) directed against both intracellular and extracellular loops of SERINC5.

Experiment

To obtain mAbs directed against SERINC5, the researchers use a prime-boost immunization strategy in mice with a full-length SERINC5.1 DNA prime and peptide boosting immunogens.

Immunogen design

Three SERINC5 peptides were selected as target epitopes in two extracellular domains (ECL1 and ECL4) and one intracellular epitope (ICL4), based on sequence, antigenicity, surface and hydrophilicity scores predicted by OptimunAntigen™. Keyhole limpet hemocyanin (KLH) was conjugated with the ECL4 and ICL4 peptides to improve immunogenicity.

Animal immunizations

BALB/c mice were immunized intradermally with codon-optimized SERINC5.1 DNA and then boosted with KLH-peptide during a 22-week immunization schedule. Mouse serum reactivity was measured by peptide ELISA. Notably, a HEK293 cell line stably transduced with tagged SERINC5.1 gene were constructed to test the specificity of the mouse sera and subsequently the mAbs. Finally based on the assessment results of peptide ELISA, whole-cell ELISA (WCE) and Western blots (WB), a group of mice were selected for cell fusion.

Hybridoma cell line fusion and sub-cloning

Hybridoma cell supernatants for reactivity to the relevant peptides were screened by peptide ELISA to select reactive clones. Next, the hybridoma supernatants and subclones were evaluated according the screening strategy using peptide ELISA, WCE and WB. Based on the analysis results, four anti- SERINC5 hybridoma cell lines were selected for mAb production. At last, these hybridomas were scaled up to produce mAbs.

Results

Four anti-SERINC5 mAbs- labeled 14C1-1, 18B6-1, 23E4-1, and 28E8-2 respectively were obtained through this prime-boost immunization strategy. The researchers then validated the specificity of the mAbs with WB analysis, and assessed the applicability for flow cytometry (FC) using the HEK293_S5.1 cell line. Through immunocytochemistry staining of HEK293_S5.1 cells and the mAbs were able to detect endogenous localization of SERINC5.1 at both the cell surface and within the cells. A virus capture assay using filtered virus stock suggest that the 14C1-1 and 23E4-1 mAbs recognize surface-exposed SERINC5 loops, and that the 28E8-2 mAb binds a more sequestered, internal loop on viral SERINC5.

"These antibodies will aid in the characterization of the functions and mechanisms of action of SERINC5 in different cell types." as the authors said.

Reference

• Molnar, Sebastian, et al. "Novel monoclonal antibodies to the SERINC5 HIV-1 restriction factor detect endogenous and virion-associated SERINC5." MAbs. Vol. 12. No. 1. Taylor & Francis, 2020.

• Endogenous studies of SERINC5, a transmembrane protein critical for restricting HIV-1 infection, finally possible

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