Notably, Wa41v-11 also resisted neutralization by mAb #33, but not by mAb #47, suggesting that mAbs #33 and #41 bind to related epitopes on VP5*

Notably, Wa41v-11 also resisted neutralization by mAb #33, but not by mAb #47, suggesting that mAbs #33 and #41 bind to related epitopes on VP5*. people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination. == INTRODUCTION == Human rotaviruses (RVs) are the leading global cause of severe diarrhea in infants and young children (1). Two effective RV vaccines, the pentavalent RotaTeq (Merck) and the monovalent Rotarix (GlaxoSmithKline), are recommended by the World Health Business for worldwide use in Macitentan young children. Other monovalent RV vaccines are licensed for use in India, Vietnam, Macitentan and China. The mechanisms by which monovalent vaccines impart heterotypic or serotype cross-reactive immunity against multiple human RV serotypes are unknown. The elucidation of these Macitentan mechanisms is critical for the development of improved, next-generation RV vaccines needed in developing countries where the efficacy of current licensed, live attenuated vaccines is lower than in developed countries (2). RVs are nonenveloped, double-stranded, segmented genome RNA viruses with a triple-layered protein (TLP) capsid composed of two surface proteins (VP4 and VP7) and a major inner protein (VP6). RVs infect mature enterocytes of the small intestine. Virus entry and infectivity are increased by proteolytic cleavage of the trimeric attachment protein VP4 to yield its stalk (VP5*) and globular head (VP8*). VP8* mediates cell attachment; VP5* facilitates cell membrane penetration. VP7 is usually a trimeric calcium-binding surface glycoprotein (3). Sequence divergence and reassortment of the genes encoding VP4 and VP7 result in great serotypic diversity worldwide among the circulating human RV Macitentan strains and a binary classification system including G (VP7) and P (VP4) serotypes and/or genotypes. In children, protection against RV contamination is mediated primarily by neutralizing antibodies (Abs) that target epitopes on VP4, VP7, or both (4). The near-atomic structure of neutralizing epitopes on VP4 and VP7 from animal and human RVs has been elucidated (58). Most isolated murine VP7-specific neutralizing monoclonal antibodies (mAbs) are serotype-specific; however, the antigenic region (1) containing amino acids 94 to 99 is usually targeted by heterotypic and homotypic neutralizing mAbs (9). VP5* is the target of heterotypic and homotypic murine mAbs; heterotypic epitopes on VP4 have been mapped to antigenic region II at amino acids 392 to 433 (11,12). In contrast, multiple murine neutralizing mAbs to VP8* appear to be primarily serotype-specific (10,13). Epidemiologic and clinical studies demonstrate that heterotypic protective immunity is usually generated after a single RV contamination (14) or immunization (15,16). The molecular basis for protective immunity after monotypic exposure is unknown. Individual anti-VP4 and anti-VP7 Ab molecules generated after monotypic RV contamination or immunization could mediate broad-based protection to new serotypes. This hypothesis is usually supported by the comparable efficacy of the monovalent Rotarix and the pentavalent RotaTeq vaccines (2). Alternatively, heterotypic immunity could be generated by an array of individual Ab molecules, each with restricted specificity against individual RV G or P type. Only one previous study examined the serotypic specificity of human anti-VP4 and anti-VP7 mAbs. In it, two heterotypic VP4 (VP8*) mAbs and a single homotypic VP7 mAb were identified (17). An additional hypothesis is usually that Abs to a shared common internal Macitentan protein epitope around the RV virion (such as VP6) may provide heterotypic protection in vivo, although they lack neutralizing activity in traditional in vitro assays (18,19). Here, we demonstrate that individual human mAbs, induced after natural infection and specific for either VP4 (primarily VP5*) or VP7, mediate potent heterotypic neutralizing activity, whereas most of the human VP8*-binding mAbs are inactive in a traditional in vitro neutralization assay. These findings reveal a molecular basis for the broad-based, serotype cross-reactive, heterotypic protection against RV observed in humans. == RESULTS == == Isolation of RV TLP-specific intestinal B cells by flow cytometry == Intestinal B cells were LAMA3 antibody isolated from adults undergoing bariatric surgery for obesity. Virtually all adults in the world have been infected with RV at least once and therefore have a notable frequency of RV-specific B cells in the intestine at constant state (20). To identify intestinal B cells that recognize RV by flow cytometry, we used purified TLPs (CDC-9, G1P[8]) labeled with Cy5 as bait. The structural integrity of the labeled TLPs was confirmed by electron microscopy (fig. S1A). The binding specificity of TLP-Cy5 to B cells expressing surface VP4- or VP7-specific immunoglobulin (Ig) was assessed using mouse hybridomas specific for VP6 and for serotypically distinct VP4s (P[8].