Immune Imprinting Drives Human Norovirus Potential for Global Spread

Abstract

Understanding the complex interactions between virus and host that drive new strain evolution is key to predicting the emergence potential of variants and informing vaccine development. Under our hypothesis, future dominant human norovirus GII.4 variants with critical antigenic properties that allow them to spread are currently circulating undetected, having diverged years earlier. Through large-scale sequencing of GII.4 surveillance samples, we identified two variants with extensive divergence within domains that mediate neutralizing antibody binding. Subsequent serological characterization of these strains using temporally resolved adult and child sera suggests that neither candidate could spread globally in adults with multiple GII.4 exposures, yet young children with minimal GII.4 exposure appear susceptible. Antigenic cartography of surveillance and outbreak sera indicates that continued population exposure to GII.4 Sydney 2012 and antigenically related variants over a 6-year period resulted in a broadening of immunity to heterogeneous GII.4 variants, including those identified here. We show that the strongest antibody responses in adults exposed to GII.4 Sydney 2012 are directed to previously circulating GII.4 viruses. Our data suggest that the broadening of antibody responses compromises establishment of strong GII.4 Sydney 2012 immunity, thereby allowing the continued persistence of GII.4 Sydney 2012 and modulating the cycle of norovirus GII.4 variant replacement. Our results indicate a cycle of norovirus GII.4 variant replacement dependent upon population immunity. Young children are susceptible to divergent variants; therefore, emergence of these strains worldwide is driven proximally by changes in adult serological immunity and distally by viral evolution that confers fitness in the context of immunity. IMPORTANCE In our model, preepidemic human norovirus variants harbor genetic diversification that translates into novel antigenic features without compromising viral fitness. Through surveillance, we identified two viruses fitting this profile, forming long branches on a phylogenetic tree. Neither evades current adult immunity, yet young children are likely susceptible. By comparing serological responses, we demonstrate that population immunity varies by age/exposure, impacting predicted susceptibility to variants. Repeat exposure to antigenically similar variants broadens antibody responses, providing immunological coverage of diverse variants but compromising response to the infecting variant, allowing continued circulation. These data indicate norovirus GII.4 variant replacement is driven distally by virus evolution and proximally by immunity in adults.

Keywords: antigenic cartography; antigenic seniority; epidemic; histo-blood group antigens; immune imprinting; neutralizing antibodies; norovirus; sequencing; surveillance; variant persistence; variants of concern.

FIG 1

Maximum likelihood tree of 930 GII.4 major capsids generated by RAxML (66). Nodes with bootstrap support of less than 70 were collapsed. Rooting was optimized by TempEst (67). Tip color indicates major capsid subtype with epidemics in red tones and GII.4 viruses that spread globally in blue tones. Two long branches are observed: one leading to Hong Kong 2019 detected on 18 March 2019 and one leading to Den Haag 2017 detected on 21 February 2017. The corresponding tips are indicated by an arrow.

FIG 2

Sequence of GII.4 blockade/neutralizing antibody antigenic sites in divergent strains compared to those in closely related strains tested here. (A) Comparison of amino acid sequences in VP1 blockade/neutralizing antibody antigenic sites in Den Haag 2017 and Hong Kong 2019 to those in Den Haag 2006 and Osaka 2007 strains studied here as VLPs. (B) Blockade/neutralizing antibody antigenic sites mapped onto the surface of Sydney 2012 P domain dimer (PDB 4wzt). Residues differing between Den Haag 2017 and 2006 or Hong Kong 2019 and Osaka 2007 are colored red in both panels A and B. Complete capsid sequences for Den Haag 2019 and Hong Kong 2019 compared to their closest sequence neighbors are shown in Fig. S5.

FIG 3

Impact of GII.4 variant evolution on ligand binding. GII.4 Osaka 2007, Hong Kong 2019, and Den Haag 2006 have typical GII.4 ligand binding patterns interacting with PGM (A) and B-type salivary ligands (B). Den Haag 2017 has lower avidity for PGM than but similar avidity for B-type saliva as the other GII.4 variants. Addition of bile stabilizes binding of Den Haag 2017 to ligands (C). Maximum binding was defined by Osaka 2007. Markers denote the mean and standard deviation from two replicates tested in two independent experiments.

FIG 4

Antigenic divergence of novel strains depends on exposure history of the population. (A to D) Blockade antibody titer in sera from children aged 1 to 2 years in 2013 to 2014 (n = 34) (A), adults in 2012 to 2014 (n = 25) (B), adults in 2017 to 2019 (n = 19) (C), and children (n = 17) after their first GII.4 (Sydney 2012) infection in 2018 to 2019 (D). Marker, individual response. Line, geometric mean titer. Error bars, 95% confidence intervals. *, P ≤ 0.03; **, P ≤ 0.0021; ****, P < 0.0001, Wilcoxon matched-pairs signed-rank test, compared to Sydney 2012 or between the two Den Haag VLPs or Osaka 2007 and Hong Kong 2019. (E to H) Antigenic cartography analysis across all serum sets, divided into separate panels for clarity: children in 2013 to 2014 (E), adults in 2012 to 2014 (F), adults in 2017 to 2019 (G), and children in 2018 to 2019 (H). Virus variants are represented as distinct black shapes, and each serological data set is represented by distinct colors and with data points encapsulated in a shaded polygon. The mean of the serological points for each serum set is indicated by distinct dark gray shapes. One grid box corresponds to a 2-fold change in titer. Note that the orientation of the plots does not matter.

FIG 5

Antigenic divergence measured by convalescent-phase outbreak sera. (A to C) Blockade antibody titer in adult sera collected from GII.4 norovirus outbreaks between 1988 and 1999 (n = 27) (A), Den Haag 2006 outbreaks in 2006 to 2011 (n = 14) (B), and Sydney outbreaks in 2012 to 2014 (n = 14) (C) were compared to time-ordered GII.4 variants. Marker, individual response. Line, geometric mean titer. Error bars, 95% confidence intervals. *, P ≤ 0.03; **, P ≤ 0.0021; ***, P ≤ 0.0002; ****, P < 0.0001, Wilcoxon matched-pairs signed-rank test, compared to US95/96 (A), Den Haag 2006 (B), or Sydney 2012 (C). (D to F) Antigenic cartography analysis across all serum sets, divided into separate panels for clarity: pre-Farmington Hills sera (D), Den Haag 2006 sera (E), or Sydney 2012 sera (F). Viral variants are represented as distinct black shapes, and each serological data set is represented by distinct colors and is encapsulated in a shaded polygon. The mean of the serological points for each serum set is indicated by distinct dark gray shapes. One grid box corresponds to a 2-fold change in titer. Note that the orientation of the plots does not matter.