Lisa C. Lindesmith; Martina Beltramello; Eric F. Donaldson; Davide Corti; Jesica Swanstrom; Kari Debbink; Antonio Lanzavecchia; Ralph S. Baric

“ Robust approaches exist for the isolation of human monoclonal antibodies that are elicited following virus infection. Using human PBMCs as a source of memory B cells, we created a panel of human mAbs directed against GII.4 strains and compared the reactivity of these mAbs to a panel of time-ordered GII.4 VLPs using EIAs and surrogate neutralization assays. We identified one novel, broadly cross reactive antibody that differentially blocks GII.4.1987 through 2009 VLP interactions with carbohydrate ligands, a potential immunotherapeutic for the treatment of acute or chronic GII.4 disease. ”

“ Noting that blockade assays are not true measurements of neutralization, NVB 71.4 has potential as a therapeutic reagent based on its broad GII.4 blockade potential and the fact that it is by nature a human antibody. Clearly, the effectiveness of NVB 71.4 at preventing or treating illness can only be determined empirically. Although one of the blockade-epitope specific mAbs with lower EC50 values/steeper Hill constants may be more effective at select strain neutralization, the breadth of strains neutralized is likely to be limited for these mAbs. ”

“ Given the acute clinical disease window, it is less likely that therapeutic antibodies will provide relief in those individuals experiencing acute infections, however, therapeutic antibodies may offer opportunities for ameliorating symptomatic disease in chronic infections. The discovery of broadly cross reactive and cross blockade human GII.4 mAbs dictates the need for a new approach to map epitopes. Our current experimental approach was designed to identify GII.4 epitopes that change over time and provide insight into broadly conserved epitope locations. ”

“ Historically, the role of IgG in norovirus protection has been unclear. By adulthood, >90% of the population [40] is positive for anti-norovirus IgG, and norovirus strains within a genogroup share a high degree of antigenic cross-reactivity as measured by EIA [34] , [51] . These facts likely skew functional interpretations of the role of serum IgG titers on susceptibility to infection and/or infection outcomes. ”

“ Previously, these residues have been implicated in regulating norovirus VLP-carbohydrate ligand binding interactions [8] , [36] , [33] . The identification of Epitope D as a human antibody blockade epitope that changes over time provides direct support for our previous hypothesis that escape from protective herd immunity may drive changes in carbohydrate ligand binding affinities over time and potential retargeting of virus infection in different human populations [8] , [31] . ”

“ Immunotherapeutics are especially needed for treating immunosuppressed populations experiencing long-term infections with chronic diarrhea. The lack of understanding of the extensive antigenic relationships among the large number of norovirus strains and the complex relationship between host protective immunity and virus antigenic heterogeneity are the primary obstacles to norovirus vaccine development. ”

“ Here we have expanded these observations using human anti-GII.4 mAbs isolated from a healthy adult donor, who has likely experienced multiple norovirus infections throughout his/her lifetime. The identification of highly significant, varying antigenic epitopes that influence VLP-carbohydrate ligand interaction provides important new insights into vaccine design and the development of therapeutics that target norovirus virions. ”

“ For example, these antibodies represent the first anti-norovirus human mAbs to be characterized, and they confirm findings from studies using mouse mAbs supporting antigenic drift and its linkage with varying carbohydrate ligand binding profiles within the GII.4 noroviruses. Further, we have demonstrated that the GII.4 NoV varying epitopes can be exchanged between time-ordered VLPs, providing a robust platform for expanding the antigenic and blockade cross reactivity of future vaccine candidates. ”

“ In addition to VLP-PGM interaction blockade assay, human mAbs were also tested for blockade of VLP-synthetic biotinylated HBGA (Bi-HBGA) interaction and ability to block VLP hemagglutination of O+ RBCs. Regardless of substrate (PGM or Bi-HBGA) , the dose-response profiles for all blockade antibodies and VLPs were similar (compare Figures 3–6 to Figure 7) . ”

“ No blockade epitopes were found to be in common between GII.4.1987 and GII.4.2006.
Prior to this work, we and others had predicted GII.4 antibody epitopes using bioinformatic techniques. As expected, discreet amino acids are repeatedly identified as potential evolving epitopes. In particular, residues 296–298 and 393–395 are consistently identified as putative epitopes that change between epidemic GII.4 strains. Additional surface residues at 333, 340, 356, 368, 372, 407 and 412–413 were also predicted as potential antibody epitopes [8] , [10] , [30] , [22] , [16] , [31] – [32] . ”

“ These data absolutely underscore the critical importance of determining the structures of several of these human mAbs with their appropriate GII.4 VLP epitopes by either cryoEM or crystallography, for informing targeted mutagenesis to identify the role of key residues in regulating antigenicity and antibody escape. ”

“ In addition to the clinical applications of therapy and diagnosis, monoclonal antibodies have also proven to be superior tools for studying viral antigenicity, evolution, and for the treatment of acute viral disease in humans [8] , [24] , [47] , [48] . ”

“ These data indicate that it is the amount of human mAb that is directly blocking the VLP from binding to PGM. Of note, VLP concentrations in blockade assays are in the low nanomolar range and therefore cannot discriminate between antibodies with sub-nanomolar affinities. ”

“ Supporting the sensitivity of epitopes to the local environment, mAbs that recognized Epitope A differentiated between GII.4.2006 VLPs and GII.4.2006 P dimers. P protein (P dimer) is a dimeric, truncated form of the major capsid protein composed of residues 214–539 [53] . P-dimers have been widely used to determine the crystallographic structure of NoV-HBGA interactions and are considered accurate reflections of the VLP surface topology [39] , [36] , [54] . P –particles can assemble as higher ordered structures composed of varying copies of the P dimer [37] , [55] . ”

“ Serum hemagglutination inhibition activity correlates with protection from gastroenteritis in persons infected with Norwalk virus. Clin Vaccine Immunol. 2012;19:284–287.22190401↑ Bull RA, Eden JS, Rawlinson WD, White PA. Rapid evolution of pandemic noroviruses of the GII.4 lineage. PLoS Pathog. 2010;6:e1000831.203609721 2 3 4 Donaldson EF, Lindesmith LC, Lobue AD, Baric RS. Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. ”

“ Sigmoidal curves were fit to the mean percent control binding calculated by comparing the amount of VLP bound to PGM in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard error of the mean. Panel B: Mean EC50 for blockade of each blocked VLP. * indicates VLPs with significantly different EC50 compared GII.4.2006.Summary of exchange mutant VLPs containing blockade Epitopes A and D and the reactivity pattern of the human mAbs that recognize these epitopes. ”

“ EIA Reactivity of plasma collected from healthy donors against norovirus VLPs.VLP-specific IgG titers in 63 plasma samples collected in early 2009 were measured by EIA using a panel of norovirus VLPs as antigen. Reciprocal ED50 dilutions (see Materials and Methods) are shown. Highlighted in red is the plasma sample of the donor NVB selected for the isolation of human mAbs. ED50 values below 10 were scored as negative and assigned a value of 1. The line shows the geometric mean value.Characterization of donor NVB plasma blockade of norovirus VLPs.Panel A: PGM binding blockade activity. ”

“ Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization. PLoS Pathog. 2008;4:e1000060.18464894↑ Lopman B, Vennema H, Kohli E, Pothier P, Sanchez A, et al. Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant. Lancet. 2004;363:682–688.15001325↑ Zhu Z, Dimitrov AS, Chakraborti S, Dimitrova D, Xiao X, et al. Development of human monoclonal antibodies against diseases caused by emerging and biodefense-related viruses. ”

“ Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors. Nature. 2008;455:532–536.187166251 2 Tan M, Hegde RS, Jiang X. The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors. J Virol. 2004;78:6233–6242.15163716↑ Choi JM, Hutson AM, Estes MK, Prasad BV. Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus. ”

“ EIA Reactivity of mAbs to GII.4.2006 VLPs and P proteins.Increasing concentrations of GII.4.2006 VLP (Panel A) or P protein (Panel B) were assayed for reactivity to the hu mAbs by EIA. Arrow indicates 1 μg/ml, the concentration of GII.4.2006 VLP scored as postive by EIA (Figure S2 and Table 2) . Bars are SEM. Predicting putative GII.4 evolving antibody epitopes
The evolution of the GII.4 noroviruses was assessed over a 36-year period of time by comparing strains from 1974 to 2010. ”

“ Euro Surveill. 2008;13↑ Vega E BL, Gregoricus N, Williams K, Lee D, Vinjé J. Novel surveillance network for norovirus gastroenteritis outbreaks, United States. Emerg Infect Dis. 2011;17:1389–1395.218016141 2 Allen DJ, Gray JJ, Gallimore CI, Xerry J, Iturriza-Gomara M. Analysis of amino acid variation in the P2 domain of the GII-4 norovirus VP1 protein reveals putative variant-specific epitopes. PLoS ONE. 2008;3:e1485.182133931 2 3 4 Cannon JL, Lindesmith LC, Donaldson EF, Saxe L, Baric RS, et al. Herd immunity to GII.4 noroviruses is supported by outbreak patient sera. ”