Population Genomics of GII.4 Noroviruses Reveal Complex Diversification and New Antigenic Sites Involved in the Emergence of Pandemic Strains

Abstract

(max 250) 14Word count in Importance: 110 (max 150) 15 Word count in Main Text: 5698 16Abstract 25 26 GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially 27 explained by the continuous emergence of antigenically distinct variants. To gain insights on the 28 mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed 29 large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 30 noroviruses exhibited a periodic replacement of predominant variants with accumulation of 31 amino acid substitutions. Genomic analyses revealed (i) a large number (87%) of conserved 32 residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) 33 variable residues that map outside of the antigenic sites. Residues from the third pattern formed 34 motifs on the surface of VP1, which suggested extensions of previously predicted and new 35 uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic make-up of the 36 GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking 37 assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the 38 circulation patterns of GII.4 variants, with two of them (C and G) containing residues (352, 357, 39 378) linked with the emergence of new GII.4 variants. Notably, the emergence of each variant 40 was followed by a stochastic diversification with minimal changes at the antigenic sites that did 41 not progress towards the next variant. This study provides a methodological framework for 42 antigenic characterization of viruses, and expands our understanding of the dynamics of GII.4 43 noroviruses that could facilitate the design of cross-reactive vaccines. 44 3 Importance 45 46 Noroviruses are an important cause of viral gastroenteritis around the world. An obstacle 47delaying the development of norovirus vaccines is an inadequate understanding of the role of 48 norovirus diversity in immunity. Using a population genomics approach, we identified new 49 residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, 50 that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful 51 monitoring of the substitutions in those residues involved in the diversification and emergence of 52 new viruses could help in the early detection of future novel variants with pandemic potential. 53Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus 54 vaccine design. 55 56

Understanding the relationship between norovirus diversity and immunity

“…Thus, antigenic site A consists of residues 294-298, 368, 372, 373; antigenic site C of 339-341, 375-378; antigenic site D of 393-397; antigenic site E of 407, 411-414; and antigenic site G of 352, 355-357, 359, 364 (Figure 1, panel D). [66][67][68][69][70] Most of these sites are on the loops and/or outer surface of the P2 subdomain where they can interact with neutralizing antibodies and attachment factors. Notably, although the overall structure of the capsid is mostly conserved between the different variants of GII.4, the length varies among genotypes.…”

“…Changes on these antigenic sites, particularly antigenic sites A, C, and G, also correlate with the circulation patterns of GII.4 variants (Figure 1, panel E, right). 70 Notably, while mutations in single antigenic sites can abolish mAbs binding, 69,70 largescale antigenic studies suggest that multiple sites are required for antigenic diversification of GII.4. 38 Indeed, structural analyses identified mAbs with footprint spanning multiple antigenic sites, suggesting that the virus may need to change multiple sites to evade the host immune response.…”

“…69 Similarly, using polyclonal guinea pig sera, our group showed that antigenic sites A and G from the GII.4 Sydney/2012 variant were both important immunodominant blockade sites. 70 Whether these immunodominant sites can induce cross-protective antibodies against multiple strains or if these antibodies are variant-specific remains to be determined. For example, as antigenic site G is more conserved compared to the other major antigenic sites/motifs, antibodies targeting site G could potentially provide protection against multiple GII.4 variants.…”

“…Dataset includes sequences collected from 1995 to 2016, as described in Tohma et al, 2019. 70 Entropy values were calculated using the Shannon Entropy-One tool, as implemented in Los Alamos National Laboratory (www.hiv.lanl.gov) for six major GII.4 variants. The boxplot shows mean and standard deviation from each antigenic and Although humans can be infected by different noroviruses (>30 genotypes, mainly from Genogroups I and II), viruses from the GII.4 genotype are the major cause of large epidemics worldwide.…”

Capturing noroviruses circulating in the population: sewage surveillance in Guangdong, China (2013-2018)

“…The novel GII.4 variant has emerged every 2-5 years and presented amino acid changes in five antigenic sites (7) associated with viral escape from herd immunity (8,9). This epochal evolution pattern is regarded as the principal mechanism adopted by GII.4 to sustain its predominance and has led to successive pandemics in the global population (10)(11)(12).…”