Structure-Based Design of Hepatitis C Virus E2 Glycoprotein Improves Serum Binding and Cross-Neutralization

ABSTRACT An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV. IMPORTANCE Hepatitis C virus infects approximately 1% of the world’s population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of bNAbs in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.

Determination of the immunogenic region in the LipL32 protein of Leptospira

Leptospirosis is a zoonotic disease caused by the pathogenic species of Leptospira. The initial symptoms include fever, myalgia, nausea, skin rash, chills, and headache, which can be misdiagnosed. LipL32 is the highly conserved and abundant outer membrane protein (OMP) of Leptospira, which is used as an antigen in serodiagnostic assays. We used three in silico methods to predict the immunodominant regions in the full-length LipL32 protein. We identified three regions, namely the N-terminus (NrLipL32, amino acid sequence 20th-120th), intermediate (amino acid sequence 120th-150th), and C-terminus (CrLipL32, amino acid sequence 160th-260th) regions. The full-length protein and two larger fragments were cloned into the pET22b plasmid and expressed in Escherichia coli BL21 (DE3). The purified proteins were used as antigens in an ELISA to detect Leptospira-specific antibodies. The CrLipL32 ELISA showed the highest sensitivity for IgM (73.3%) and IgG (65%), followed by the full-length rLipL32 ELISA (IgM 68% and IgG 60%). The full-length rLipL32 ELISA showed high specificity (IgM 85% and IgG 75%), followed by the NrLipL32 ELISA (IgM 75% and IgG 60%). The intermediate fragment showed very low sensitivity (IgM 17% and IgG 2%). The sensitivity of the rLipL32 ELISA could be enhanced by adding other OMPs of Leptospira.