Variation of surface antigen expression is a mechanism used by microbes to adapt to and persist within their host habitats. Helicobacter pylori, a persistent bacterial colonizer of the human stomach, can alter its surface Lewis (Le) antigen expression. We examined H. pylori colonization in mice to test the hypothesis that host phenotype selects for H. pylori (Le) phenotypes. When wild-type and Leb-expressing transgenic FVB/N mice were challenged with H. pylori strain HP1, expressing Lex and Ley, we found that bacterial populations recovered after 8 mo from Leb-transgenic, but not wild-type, mice expressed Leb. Changes in Le phenotype were linked to variation of a putative galactosyltransferase gene (β-(1,3)galT); mutagenesis and complementation revealed its essential role in type I antigen expression. These studies indicate that H. pylori evolves to resemble the host's gastric Le phenotype, and reveal a bacterial genetic locus that is subject to host-driven selection pressure.
We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/J H (H3J) fragments. One IGHV gene provided a universal V H scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal V H scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated K D values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.
ARTICLE HISTORY
SUMMARY
During infection, humoral immunity produces a polyclonal response with various immunoglobulins recognizing different epitopes within the microbe or toxin. Despite this diverse response, the biological activity of an antibody (Ab) is usually assessed by the action of a monoclonal population. We demonstrate that a combination of monoclonal antibodies (mAbs) that are individually disease-enhancing or neutralizing to Bacillus anthracis protective antigen (PA), a component of anthrax toxin, results in significantly augmented protection against the toxin. This boosted protection is Fc gamma receptor (FcγR)-dependent and involves the formation of stoichiometrically defined mAb-PA complexes that requires immunoglobulin bivalence and simultaneous interaction between PA and the two mAbs. The formation of these mAb-PA complexes inhibits PA oligomerization, resulting in protection. These data suggest that functional assessments of single Abs may inaccurately predict how the same Abs will operate in polyclonal preparations and imply that potentially therapeutic mAbs may be overlooked in single Ab screens.
Results indicated that L. monocytogenes virulence genes underwent positive selection that is consistent with the diversification of 2 evolutionary lineages: lineage I is clonal and associated with encephalitis, and lineage II is more genetically diverse and equally likely to cause both major forms of listeriosis in cattle.
Background: Ig , ␥, and ␣ constant regions can affect Ag specificity, but this is unknown for IgE. Results: IgE and IgA variable regions differ in specificity and cleavage rates compared with each other and IgG subclasses. Conclusion: Like IgG subclasses, the IgA and IgE constant regions can affect Ag binding specificities. Significance: These results extend the principle that the constant region can affect variable region specificity to IgE.
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