Abstract
The mechanisms by which TLR4-based adjuvants enhance immunogenicity are not fully understood. We have taken advantage of a novel knock-in mouse strain that homozygously expresses two single-nucleotide polymorphisms (SNPs) that are homologous to human TLR4 (rs4986790 and rs4986791) and have been associated with LPS hyporesponsiveness in vivo and in vitro. TLR4-SNP (coexpressing mutations D298G/N397I in TLR4) mice that recapitulate the human phenotype were compared with wild-type (WT) mice for their hapten-specific Ab responses after immunization with hapten 4-hydroxy-3-nitrophenyl acetyl (NP) NP-Ficoll or NP-OVA in the absence or presence of a water-soluble TLR4 analog adjuvant, E6020. IgM and IgG anti-NP responses were comparable in WT and TLR4-SNP mice after immunization with either NP-Ficoll or NP-OVA only. E6020 significantly yet transiently improved the IgM and IgG anti-NP responses of both WT and TLR4-SNP mice to NP-Ficoll (T-independent), with modestly enhanced Ab production in WT mice. In contrast, T-dependent (NP-OVA), adjuvant-enhanced responses showed sustained elevation of NP-specific Ab titers in WT mice, intermediate responses in TLR4-SNP mice, and negligible enhancement in TLR4-/- mice. E6020-enhanced early humoral responses in WT and TLR4-SNP mice to NP-OVA favored an IgG1 response. After a second immunization, however, the immune responses of TLR4-SNP mice remained IgG1 dominant, whereas WT mice reimmunized with NP-OVA and E6020 exhibited increased anti-NP IgG2c titers and a sustained increase in the IgG1 and IgG2c production by splenocytes. These findings indicate that E6020 increases and sustains Ab titers and promotes isotype class switching, as evidenced by reduced titers and IgG1-dominant immune responses in mice with TLR4 insufficiency.