This initial 1:5 dilution was then serially diluted two-fold in 100 L volumes for an additional five dilutions

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This initial 1:5 dilution was then serially diluted two-fold in 100 L volumes for an additional five dilutions. while fraction 9 represents the bottom of the gradient with the highest sucrose concentration. Note the increased proportion of gD-RIC in fractions 4-9 compared all other constructs. The leftmost BPTU lane contains protein size markers. Image_2.tiff (1.7M) GUID:?5AC0260A-70A9-41D2-9422-35E7722971B0 Supplementary Figure?3: Antibody Response to the Human IgG1 RIC Backbone ELISA measuring antibody responses using serially diluted (1:100 to 1 1:10,000) dose 3 serum from mice vaccinated with gD-RIC or PBS. Serum binding was measured using HL, 6D8 antibody with heavy and light chains; He, 6D8 heavy chain with epitope tag; or HLe-gD, gD-RIC made up of 6D8, epitope tag, and gD. Representative results from two impartial experiments are given as mean OD450 values from two samples standard error. Image_3.jpeg (62K) GUID:?6B8EB56E-76D5-4798-9FAB-5175CBE20999 Data Availability StatementThe original contributions presented in the study are publicly available. The gD-RIC construct information is deposited into the GenBank repository, accession OP819560. Abstract Introduction It has been known for over half a century that mixing an antigen with its cognate antibody in an immune BPTU complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural contamination. Materials and methods In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. Results gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as BPTU well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. Discussion This work demonstrates that this RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform. Keywords: herpes simplex virus (HSV), vaccine, immune complex (IC), plant-made, complement receptor c1q, neutralizing antibodies, glycoprotein D (gD), IgG fusion Highlights Self-binding immune complexes increase immune receptor C1q binding 25-fold over traditional immune complexes. Antigen-specific antibody titers increased by up to 1 1,000-fold using self-binding immune complexes compared to traditional immune complexes. Improved neutralizing and cross-neutralizing antibodies against HSV-2 and HSV-1. 1.?Introduction Antibodies are one of the most widely produced therapeutic brokers, comprising the largest share of the global biopharmaceutical market. In 2021, the one-hundredth antibody therapy was approved by the FDA (1). While antibodies by themselves are highly useful, it is becoming increasingly common to fuse antibodies to other proteins of interest to imbue them with desirable properties. Fusion to IgG Rabbit polyclonal to ANGPTL7 antibody often provides enhanced solubility and stability of the fusion partner due to the inherent stability of IgG molecules and allows simple and highly efficient purification via protein A/G affinity chromatography (2). Additionally, IgG fusions may have extended serum half-life, as IgG are guarded from degradation in endosomes due to their ability to bind neonatal.