The Antigenic Topology of Norovirus as Defined by B and T Cell Epitope Mapping: Implications for Universal Vaccines and Therapeutics

Figures

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  Figure 1. (A) Organization of the human norovirus genome. ORF1 (green) encodes the nonstructural proteins, ORF2 (purple) encodes the major structural capsid protein, VP1, and ORF3 (blue), encodes the minor structural protein, VP2. Amino acids are numbered according to a representative GI.1 genome (GenBank: KF429765.1). The VP1 protein is divided into two major domains: Shell (S) and Protruding (P) [17]. The S domain is immediately preceded by a short N-terminal arm (NTA), and the S and P domains are connected by a flexible hinge region (H). The P domain is further subdivided into P1 and P2. (B) Structural modeling of the capsid VP1 dimer. VP1 dimerizes from two protein chains (A and B), with the S domain constructing the inner part of the capsid that surrounds the RNA genome. P1 domains fold into each other, allowing P2 to become the most exposed portion of the dimer. Loops are labeled according to GI.1 nomenclature [18]. ChimeraX was used to model loops and HBGA binding (GI.1 PDB 2ZL6). (C) Genetic diversity of norovirus VP1. Representative VP1 sequences from each genotype were aligned using MEGA. Genotypes cluster into Genogroups GI through GVII. The majority of human noroviruses belong…

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  Figure 2. – Overview of proposed adaptive immune responses to murine norovirus infection in vivo. Movement of virus or virus-infected cells is marked by red dashed lines with arrows. B cell- (blue) and T cell- (green) related movement/cytokine or immunoglobulin secretion are marked by blue and green dashed lines, respectively. Maturation of memory B and T cells is marked by solid lines. Acute MNV infection begins with endocytosis at M cells found in Peyer’s patches of the intestinal tract [47]. Following entry into the lamina propria, MNV infects CD300lf-positive macrophages (Mθ), dendritic cells (DCs), B cells, and T cells [46,48,49,50]. Innate immune responses are mediated through the production and release of interferons (IFNs), which aid in the activation of the adaptive immune response. Professional antigen-presenting cells (APCs) can recognize antibody-tagged virus via Fc receptors as part of phagocytosis. Additionally, APCs can migrate to mesenteric lymph nodes (MLNs). Presentation of MNV peptides on MHC class I molecules leads to the stimulation of Th1 proinflammatory responses. CD4+ Th1 cells release various cytokines that upregulate the activity of CD8+ cytotoxic T lymphocytes…

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  Figure 3. Monoclonal antibody types and their functional characterization. (A) Types of monoclonal antibodies used to map norovirus epitopes are shown. Intact immunoglobulins derived from traditional hybridoma technology have been used extensively. Various forms of Fab fragments can be isolated from Fc regions through peptide digestion of intact immunoglobulins. Recombinant antibody technology based on the cloning and engineering of selected complementarity-determining regions (CDRs) in various immunoglobulin scaffolds have gained increasing use. Single-chain variable fragments (scFvs) are created by expression of the single heavy and light chain sequences of a complete antigen-binding site joined by a flexible linker. Variable heavy chain (VHH) antibodies isolated from camelids have proven useful for mapping norovirus epitopes due to their small size and accessibility to hidden epitopes. (B) Commonly used assays to assess antibody function are shown. Surrogate neutralization assays (HAI and HBGA blockade) measure the ability of antibodies to interfere with the binding of HuNoV VLPs to blood group carbohydrates. Positive HAI activity is scored by the ability of antibodies to prevent blood…

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  Figure 4. Comparison of HBGA blockade epitopes mapped to (A) GI.1, (B) GII.4, and (C) GII.10 P domains. HBGA binding residues are denoted in gold above the linear representation and the three-dimensional models of HuNoV P domains. Adjacent tables record epitope specificities and coloring that corresponds to the positions of the amino acids on both the linear and three-dimensional diagrams. Antibodies marked with an asterisk (*) do not have an antibody/virus structure associated with their epitope definition. ChimeraX was used to model amino acid binding sites (GI.1 PDB 2ZL6, GII.4 PDB 2OBS, GII.10 PDB 3ONU).

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  Figure 5. Relationship of two neutralizing B cell epitopes to receptor or HBGA binding sites. (A) MNV neutralizing epitope mapping to the P2 domain. Neutralizing mAb A6.2 binds to a conformational epitope (purple) that overlaps the CD300lf receptor binding domain (gold). (B) GII.4 neutralizing epitope mapping to the P2 domain. Neutralizing mAb 10E9 binds to an epitope that spans the P2 strains [65]. 2D3 is predicted to recognize some of the same residues as MAb A6.2 and possibly overlap with the CD300lf binding site [172]. Interestingly, resistance mutations to 2D3 took about 20 passages to become detectable, which suggested that the 2D3 mAb recognized critical residues involved in receptor binding [65]. Amino acid changes that resulted in loss of neutralization for both MAb A6.2 and 2D3 forced the P domain into an unfavorable conformation, instead of altering the dimer interface (orange). HBGA binding residues are highlighted in gold. Adjacent tables record epitope specificities and coloring that corresponds to the positions of the amino acids on both the linear and three-dimensional diagrams. ChimeraX was used to model amino acid binding sites (MNV PDB 6C6Q, GII.4 PDB 6EWB).

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  Figure 6. Summary of T cell epitopes mapping to the norovirus VP1 sequence. T cells were isolated from mice and humans with either prior norovirus infection or immunization with VLPs. T cells were stimulated with peptides spanning a norovirus genotype of interest. Reactive epitopes are recorded in the table and illustrated on the linear schematic of VP1. MNV-specific epitopes are shaded grey, GI genotype-specific epitopes are shaded red, and GII genotype-specific epitopes are shaded blue. Three cross-genotype reactive epitopes (bolded in the table), including a human-derived T cell epitope mapping in the S domain (outlined in green), are shaded in gold and modeled on the 3D structure of GI.1 NV VP1. Taken together, the majority of T cell epitopes have been mapped to the S and P1 domains of VP1.