Low antigen dose favours selection of somatic mutants with hallmarks of antibody affinity maturation

The immunization schedule is critical for the derivation of high‐affinity antibodies, low antigen dose being particularly favourable for the development of a more efficient memory response. To analyse the molecular events underpinning this preference, we analysed the early maturation of the response to the hapten 2‐phenyloxazolone (phOx) using low and high doses of immunogen. The phOx response is initially dominated by antibodies expressing the VkOx1‐Jk5 light chain and the hallmark of the early stages of maturation is the substitution of His 34 by Asn or Gln increasing affinity 10‐ or eightfold, respectively, and of Tyr 36 by Phe. High‐affinity antibodies express mutations at both sites. We cloned and sequenced VkOx1‐Jk5 light chains from antigen‐specific B cells taken 14 and 21 days after immunization with high and low antigen doses. We found that overall, the derived sequences were more mutated both at longer times and at higher dose. At day 14, His 34 was more frequently mutated at the higher than at the lower dose, while at day 21 the reverse was true. On the other hand, the His 34/Tyr 36 mutation pair was more frequent at low than high doses at both 14 and 21 days. Furthermore, at both times, the low immunization protocol yielded double mutants in cells with a lower mutation background. It appears therefore that while the higher dose may favour the acquisition of individual critical mutations, low‐dose immunization favours the selection of a more focused mutational pattern, whereby advantageous mutations are associated with a low mutational background.