Expansion and Refinement of Deep Sequence-Coupled Biopanning Technology for Epitope-Specific Antibody Responses in Human Serum

Abstract

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses. Here, we describe refinements that expand the number of patient samples that can be processed at one time, increasing the utility of this technology for rapidly responding to emerging infectious diseases. We used dengue virus (DENV) as a model system since much is already known about the antibody response. Sera from primary DENV-infected patients (n = 28) were used to pan an MS2 bacteriophage VLP library displaying all possible 10-amino-acid peptides from the DENV polypeptide. Selected VLPs were identified by deep sequencing and further investigated by enzyme-linked immunosorbent assay. We identified previously described immunodominant regions of envelope and nonstructural protein-1, as well as a number of other epitopes. Our refinement of the deep sequence-coupled biopanning technology expands the utility of this approach for rapidly investigating the specificity of antibody responses to infectious diseases.

Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

“…DENV-infected patients make antibodies to this region of NS1, and these antibodies have been implicated in the binding of platelets and impacting coagulation pathways [ 47 , 49 ]. Interestingly, we have identified antibodies against a number of other highly conserved sequences selected for this study in DENV-infected patient sera including aa 56–71, 154–170, and 193–204 using a deep-sequencing method [ 50 ]. Identification of antibodies against these regions of NS1, along with the highly conserved sequences among the four serotypes, gives precedent for the importance of these regions in protecting against DENV disease.…”

“…In our previous research, we utilized our deep sequence-coupled biopanning method (DSCB) to identify regions of the DENV genome in which patients made antibodies against [ 37 , 50 ]. The envelope (E) protein, specifically the Fusion Loop on the envelope protein, was a particularly immunodominant region; a result that has been observed in other studies [ 53 , 54 , 55 , 56 , 57 ].…”

“…In our previous research, the tail region of NS1 (aa 325–337) was highly selected by individuals who had been infected with their first DENV infection [ 50 ]. Additionally, antibodies from patients with secondary DENV infection had a binding profile similar to that of primary infected DENV patients, when sera were tested via ELISA [ 50 ].…”

“…In our previous research, the tail region of NS1 (aa 325–337) was highly selected by individuals who had been infected with their first DENV infection [ 50 ]. Additionally, antibodies from patients with secondary DENV infection had a binding profile similar to that of primary infected DENV patients, when sera were tested via ELISA [ 50 ]. However, our results provided in the current study show that the immunization against the NS1 tail region (aa 325–337) elicited high antibody titers to the cognate peptide, but these antibodies insufficiently bound to soluble NS1 and DENV-infected cells.…”

“…Additionally, our peptide ELISAs test the binding of sera antibodies to the linear peptides on the surface of the plate. However, in our previous work the peptide ELISAs testing human sera against the NS1 tail peptide were testing sera antibodies in the same way [ 50 ]. This indicates that humans naturally infected with primary and secondary DENV infections made antibodies that were capable of binding to the linear form of the NS1 tail epitope.…”