The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

SummaryThe acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST + ) and uninfected TST À ) with M. tuberculosis. From a total of 22 V genes analysed, the TST À population preferred the V H 3-23 and Vj1 genes. The V H 3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST À population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST + population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST À population.The antibodyome difference between both populations suggests a preference for antibodies with V H 3-23, D3-3, J H 4 gene usage by the TST À population that could be associated with resistance to infection with M. tuberculosis.

Streptomyces spp. are bacteria that are responsible for the degradation of aromatic compounds and produce secondary metabolites. Here, we present a complete genome sequence of Streptomyces sp. strain S6, which was isolated from an oil palm plantation, with a 7.8-Mbp liner chromosome, a GC content of 72%, and 4,266 coding sequences.