Role of Memory B Cells in Hemagglutinin-Specific Antibody Production Following Human Influenza A Virus Infection.

When influenza A virus infects an immune individual, preexisting memory B cell (MBC) activation and rapid anamnestic antibody production plays a key role in viral clearance. The most effective neutralizing antibodies target the antigenically variable head of the viral hemagglutinin (HA); antibodies against the conserved HA stalk provide broader but less potent protection. In this review, we provide a comprehensive picture of an adult's HA-specific antibody response to influenza virus infection. The process is followed from preexisting HA-specific MBC activation and rapid production of anti-HA antibodies, through to germinal center seeding and adaptation of the response to novel features of the HA. A major focus of the review is the role of competition between preexisting MBCs in determining the character of the HA-reactive antibody response. HA novelty modifies this competition and can shift the response from the immunodominant head to the stalk. We suggest that antibodies resulting from preexisting MBC activation are important regulators of anti-HA antibody production and play a role in positive selection of germinal center B cells reactive to novel HA epitopes. Our review also considers the role of MBCs in the effects of early-life imprinting on HA head- and stalk-specific antibody responses to influenza infection. An understanding of the processes described in this review will guide development of vaccination strategies that provide broadly effective protection.

Broad Hemagglutinin-Specific Memory B Cell Expansion by Seasonal Influenza Virus Infection Reflects Early-Life Imprinting and Adaptation to the Infecting Virus.

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel ß-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

Studies on the Chitin Binding Property of Novel Cysteine-Rich Peptides from Alternanthera sessilis.

Hevein-like peptides make up a family of cysteine-rich peptides (CRPs) and play a role in plants in their defense against insects and fungal pathogens. In this study, we report the isolation and characterization of six hevein-like peptides, aSG1-G3 and aSR1-R3, collectively named altides from green and red varieties of Alternanthera sessilis, a perennial herb belonging to the Amaranthaceae family. Proteomic analysis of altides revealed they contain six cysteines (6C), seven glycines, four prolines, and a conserved chitin-binding domain (SXYGY/SXFGY). Thus far, only four 6C-hevein-like peptides have been isolated and characterized; hence, our study expands the existing library of these peptides. Nuclear magnetic resonance (NMR) study of altides showed its three disulfide bonds were arranged in a cystine knot motif. As a consequence of this disulfide arrangement, they are stable against thermal and enzymatic degradation. Gene cloning studies revealed altides contain a three-domain precursor with an endoplasmic reticulum signal peptide followed by a mature CRP domain and a short C-terminal tail. This indicates that the biosynthesis of altides is through the secretory pathway. (1)H NMR titration experiments showed that the 29-30-amino acid altides bind to chitin oligomers with dissociation constants in the micromolar range. Aromatic residues in the chitin-binding domain of altides were involved in the binding interaction. To the best of our knowledge, aSR1 is the smallest hevein-like peptide with a dissociation constant toward chitotriose comparable to those of hevein and other hevein-like peptides. Together, our study expands the existing library of 6C-hevein-like peptides and provides insights into their structure, biosynthesis, and interaction with chitin oligosaccharides.

Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

Discovery and characterization of pseudocyclic cystine-knot α-amylase inhibitors with high resistance to heat and proteolytic degradation.

Obesity and type 2 diabetes are chronic metabolic diseases, and those affected could benefit from the use of α-amylase inhibitors to manage starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3, isolated from an Apocynaceae plant show promise for further development as orally active α-amylase inhibitors. These linear peptides retain the stability known for cystine-knot peptides in the presence of harsh treatment. They are resistant to heat treatment and endopeptidase and exopeptidase degradation, which is characteristic of cyclic cystine-knot peptides. Our NMR and crystallography analysis also showed that wrightides, which are currently the smallest proteinaceous α-amylase inhibitors reported, contain the backbone-twisting cis-proline, which is preceded by a nonaromatic residue rather than a conventional aromatic residue. The modeled structure and a molecular dynamics study of Wr-AI1 in complex with yellow mealworm α-amylase suggested that, despite having a similar structure and cystine-knot fold, the knottin-type α-amylase inhibitors may bind to insect α-amylase via a different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine-knot α-amylase inhibitors and their biosynthesis in plants follow a secretory protein synthesis pathway. Together, our findings provide insights for the use of the pseudocyclic α-amylase inhibitors as useful leads for the development of orally active peptidyl bioactives, as well as an alternative scaffold for cyclic peptides for engineering metabolically stable human α-amylase inhibitors.