Day 14 post infection spleens were harvested and individually stimulated with pooled peptides (1C10) of A/CA HA. and could be leveraged to boost hAb responses. responses to new epitopes. Recent technological advances in B cell sequencing demonstrates that reactivation of memory B cell responses to drifted influenza strains result in either: 1) memory B cells that Finafloxacin undergo somatic hypermutation (SHM) and become more adapted to the drifted influenza HA antigen, or 2) the memory B cell repertoire does not adapt, and instead directly differentiates into a plasmablast to secrete antibodies that recognize the undrifted epitopes of previous strains (21). This latter phenomenon is termed original antigenic sin (OAS) and is phenotypically characterized by diminished antibody responses to novel parts of the drifted strain upon exposure to that strain and increased antibody responses to the initial pre-exposure strain (22C29). It is not clear what intrinsic or extrinsic factors influence whether memory B cells adapt to a drifted strain. One hypothesis is that the HA-specific memory B cells ability to present conserved HA-based cognate antigens via MHC Class II to preexisting HA-reactive memory CD4+ T cells initiates further adaptation by SHM of memory B cells to the drifted strain (12, 30C32). It has been shown that HA-specific B cells are solely Finafloxacin helped by HA-reactive CD4+ T cells in the mouse model of influenza (33). Therefore, we postulate that if drifted HAs contain conserved MHC II epitopes, preexisting memory CD4+ T cells can help B cells adapt to the newer influenza strain. RESULTS hAb responses to drifted recombinant HA is dependent upon mouse MHC Class II haplotype In order to Rabbit Polyclonal to DQX1 assess how preexisting immunity augments subsequent hAb responses to drifted influenza immunization, we established mouse models by first exposing C57BL/6 and CB6F1 mice to 0.5 LD50 (Lethal Dose 50%) A/CA/7/09 (A/CA), the prototype H1N1 virus from the 2009 2009 pandemic, allowing the mice to undergo infection, seroconversion, and recuperation for 28 days and then re-exposing mice to 1 1 g of the drifted strain A/PR8 HA by intramuscular immunization (i.m.). Mice were weighed daily and bled at day 21 post-primary and secondary immunization to measure both hAb titer by HAI assay and HA titer by enzyme-linked immunosorbent assay (ELISA). In our models, C57BL/6 mice preexposed to A/CA/7/09 (A/CA), display little reactivity by HAI and ELISA to A/PR/8/34 (A/PR8) 21 days after A/PR8 immunization when compared to A/CA preexposed CB6F1 mice which display 32-fold increase in HAI and ELISA responses to the drifted A/PR8 HA immunization (Figure 1A-?-D).D). Importantly, both mouse models display HA binding Finafloxacin cross-reactivity by ELISA to A/PR8 post primary infection with A/CA but do not display significant cross-reactive HAI titers, suggesting that they do not share neutralizing epitopes. To further test if the increased hAb titer to A/PR8 in CB6F1 mice resulted in enhanced protection, we then challenged all groups with 1000 LD50 of A/PR8. Unsurprisingly, protection from weight loss and lethality directly corresponded to hAb A/PR8 titer. Only the CB6F1 mice with high A/PR8 HAI titers demonstrated 100% protection from weight loss and lethality (Figure 1E-?-F).F). This finding that A/CA preexposed F1 but not B6 mice display enhanced antibody responses to immunization with a Finafloxacin drifted HA, A/PR8, led us to hypothesize this discrepancy was possibly due to mouse genetic background. Open in a separate window Figure 1: Pre-exposed CB6F1 but not C57BL/6 mice have increased antibody responses to drifted influenza immunization.C57BL/6 (n = 4 per group) and CB6F1 mice were intranasally exposed to A/CA (500 pfu/mouse) or PBS then 28 days later immunized with A/PR8 rHA (1 g/mouse). Mice were bled at 21 days post primary and secondary immunization to determine A/CA and A/PR8 HAI and ELISA titers. Mice were then challenged with 1000 LD50 A/PR8 to determine protection capability of the secondary response. A/PR8 HAI titer in C57BL/6 mice is.