Figure 3.

The LNP-adjuvanted protein vaccine induces strong antigen-specific LLPC and class-switched, somatically mutated MBC responses

(A–C) BALB/c mice received a single IM immunization with rHA or rHA mixed with eLNP or AddaVax as described in Figure 1. PR8 HA-specific LLPC and MBC responses were examined in bone marrow and spleen, respectively, 20 weeks after immunization. n = 8 mice/group. Symbols represent individual animals. Data shown are mean + SEM and are pooled from two independent experiments.

(A) Gating strategy for measuring HA-specific LLPC and MBC responses.

(B and C) PR8 HA-specific LLPC (IgD⁻Dump⁻B220⁻CD138⁺PR8 HA⁺, B) and MBC (IgD⁻Dump⁻CD138⁻B220⁺CD19⁺CD38⁺GL7⁻PR8 HA⁺, C) responses.

(D–H) Immune repertoire profiling of PR8 HA-binding MBCs. Mice received a single ID immunization with rHA formulated with eLNP or AddaVax. PR8 HA-specific MBCs were sorted from spleens 80 days after immunization.

(D) Sorting strategy.

(E) Number of PR8 HA-specific MBCs recovered from the sort.

(F) Clone counts of IgM⁻IgD⁻ HA-binding cells. Symbols represent individual animals.

(G) HA-binding clones binned by their somatic hypermutation (SHM) percentage in the IGHV gene (compared with the nearest germline gene). Each clone counts only once.

(H) IGHV gene usage in sorted HA⁺ MBCs (the heatmap indicates the fraction of clones). Red cells denote values that are between 0.21–0.59.

In (G) and (H), IGHV genes present in at least 6 mice were included. Values in parentheses denote clone numbers. In (D)–(H), data were obtained from one experiment; n = 5 per group. Statistical analysis: in (B), (C), (E), and (F), unpaired two-tailed Mann-Whitney U test was conducted. ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001. See also Figure S4.