Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.

Rapid antigenic evolution in the influenza A virus hemagglutinin precludes effective vaccination with existing vaccines. To understand this phenomenon, we passaged virus in mice immunized with influenza. Neutralizing antibodies selected mutants with single amino acid hemagglutinin substitutions that increased virus binding to cell surface glycan receptors. Passaging these high avidity-binding mutants in naïve mice, but not immune mice, selected for additional hemagglutinin substitutions that decreased cellular receptor binding avidity. Analyzing a panel of monoclonal antibody hemagglutinin escape mutants revealed a positive correlation between receptor binding avidity and escape from polyclonal antibodies. We propose that in response to variation in neutralizing antibody pressure between individuals, influenza A virus evolves by adjusting receptor binding avidity via amino acid substitutions throughout the hemagglutinin globular domain, many of which simultaneously alter antigenicity.Influenza A virus remains an important human pathogen due largely to its ability to evade antibodies specific for its attachment protein, the hemagglutinin (HA). This "antigenic drift" is due to accumulation of amino acid substitutions in HA epitopes recognized by antibodies that neutralize viral infectivity by blocking interaction of HA with sialic acid residues on hostcell membranes (1-3). The H1 subtype HA has four antigenic sites recognized by monoclonal antibodies with high neutralizing activity, designated Sa, Sb, Ca, and Cb (4). How can HA escape polyclonal antibodies given that the frequency of variants with simultaneous multiple point mutations is exceedingly low (5)? A popular model posits sequential selection by different individuals whose antibody responses focus on different individual antigenic sites (6,7).To better understand how antigenic drift occurs in human populations, we revisited classical experiments modeling drift in outbred Swiss mice (8). We generated three separate infectious stocks of the mouse-adapted strain A/Puerto Rico/8/34 (H1N1) (PR8) in MDCK cells using

We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgGproducing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.

Translational fidelity, essential for protein and cell function, requires accurate tRNA aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of 1 error per 10,000 to 100,000 couplings 1, 2. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower 3–6. Here, we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to 10-fold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands, or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage 7, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Immunodominance defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody immunodominance despite its importance to immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible immunodominance hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changes as the immune response progresses and depending on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrate “original antigenic suppression”, where antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody immunodominance to viruses and other immunogens.

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The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85␣ regulatory subunit of phosphatidylinositol 3-kinase, phospholipase C␥, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3⅐SH3 complex. In pORF3-expressing cells, pp60 src was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.Hepatitis E virus (HEV), 1 the causative agent for hepatitis E, is a waterborne pathogen endemic to much of the developing world where it causes rampant sporadic infections and large scale epidemics (1-4). While the infection is self-limited with no associated chronicity, a fraction of the patients progress to fulminant hepatitis (5, 6), the most severe form of acute hepatitis. High mortality rates of 20 -30% reported for HEV infection during pregnancy (7,8) are also the result of fulminant hepatitis. The reasons for this and the mechanisms of viral pathogenesis are not known. The studies on HEV biology and pathogenesis have been severely restricted by the lack of a reliable cell culture system and small animal models of viral infection. We have used subgenomic expression strategies to study the properties and functions of individual HEV gene products toward understanding viral replication and pathogenicity (9 -12).The HEV genome is a ϳ7.5-kilobase polyadenylated, positive-sense RNA that contains three open reading frames (ORFs) designated ORF1, ORF2, and ORF3 (13). The ORF3 of HEV encodes a protein of ϳ13.5 kDa, called pORF3, for which no function has been assigned. When expressed in animal cells, pORF3 is phosphorylated at a single serine residue (Ser 80 ) in its 123-amino acid primary sequence (11). In vitro phosphorylation experiments suggested that pORF3 may be a substrate for the mitogen-activated protein (MAP) kinase, and subcellular fractionation revealed its association with the cytoskeleton (11). Recent results using inhibitors, activators, and dominant negative alleles show that pORF3 is a substrate for the extracellular signal-regulated kinase (ERK) as well as the stressactivated pr...

Here, we address the question of why the influenza A virus hemagglutinin (HA) does not escape immunity by hyperglycosylation. Uniquely among dozens of monoclonal antibodies specific for A/Puerto Rico/8/34, escape from H28-A2 neutralization requires substitutions introducing N-linked glycosylation at residue 131 or 144 in the globular domain. This escape decreases viral binding to cellular receptors, which must be compensated for by additional substitutions in HA or neuraminidase that enable viral replication. Sequence analysis of circulating H1 influenza viruses confirms the in vivo relevance of our findings: natural occurrence of glycosylation at residue 131 is always accompanied by a compensatory mutation known to increase HA receptor avidity. In vaccinated mice challenged with WT vs. H28-A2 escape mutants, the selective advantage conferred by glycan-mediated global reduction in antigenicity is trumped by the costs of diminished receptor avidity. These findings show that, although N-linked glycosylation can broadly diminish HA antigenicity, fitness costs restrict its deployment in immune evasion.antigenic drift | viral evolution