Adjuvant activity of naturally occurring monophosphoryl lipopolysaccharide preparations from mucosa-associated bacteria

Abstract

Natural heterogeneity in the structure of the lipid A portion of lipopolysaccharide (LPS) produces differential effects on the innate immune response. Gram-negative bacterial species produce LPS structures that differ from the classic endotoxic LPS structures. These differences include hypoacylation and hypophosphorylation of the diglucosamine backbone, both differences known to decrease LPS toxicity. The effect of decreased toxicity on the adjuvant properties of many of these LPS structures has not been fully explored. Here we demonstrate that two naturally produced forms of monophosphorylated LPS, from the mucosa-associated bacteria Bacteroides thetaiotaomicron and Prevotella intermedia, function as immunological adjuvants for antigen-specific immune responses. Each form of mucosal LPS increased vaccination-initiated antigen-specific antibody titers in both quantity and quality when given simultaneously with vaccine antigen preparations. Interestingly, adjuvant effects on initial T cell clonal expansion were selective for CD4 T cells. No significant increase in CD8 T cell expansion was detected. MyD88/Toll-like receptor 4 (TLR4) and TRIF/TLR4 signaling pathways showed equally decreased signaling with the LPS forms studied here as with endotoxic LPS or detoxified monophosphorylated lipid A (MPLA). Natural monophosphorylated LPS from mucosa-associated bacteria functions as a weak but effective adjuvant for specific immune responses, with preferential effects on antibody and CD4 T cell responses over CD8 T cell responses.

Fig 1

Predominant structures of the LPS preparations used as adjuvants.

Fig 2

Antigen-specific antibody responses in sera of HBV vaccine-immunized C57BL/6 mice. (A) Groups of C57BL/6 mice were immunized s.c. with an HBV vaccine in the absence (+DMSO) or presence (3 μg SmLPS, 10 μg SmMPLA, 10 μg BtLPS, or 10 μg PiLPS) of LPS preparations on day zero, followed by a boost immunization 5 weeks after the primary immunization. Serum was sampled before and after each of the immunizations, and the titers of HBsAg-specific IgM, total IgG, IgG1, IgG2c, and IgG2b were determined by ELISA by limiting dilution. Arrowheads indicate times of vaccination. (B) Antigen-specific IgG2a titers after the third immunization with egg white albumin emulsified in incomplete Freund's adjuvant with either a vehicle control (+DMSO) or one of the adjuvant preparations. (C) Antigen-specific IgG2b titers after the second immunization with iPR8 influenza virus particles and either a vehicle control (+DMSO) or one of the adjuvant preparations. Results are expressed as means ± standard errors of the means of the antibody titer, calculated as the inverse of the dilution factor (df) needed to give the half-maximum value (max_1/2) of the absorbance curve after 10-fold serial dilutions. Asterisks indicate significant differences from the DMSO control group at a 95% (P < 0.05) confidence level, as determined using two-way ANOVA and Bonferroni post hoc analysis.

Fig 3

Short-term T cell clonal expansion is increased in the spleens of animals immunized with any of the LPS preparations. (A) Schematic of the adoptive transfer and immunization protocol used on recipient C57BL/6 (CD45.2) mice. Spleen cells (10⁵ OTI.SJL cells and 1.5 × 10⁵ OTII.SJL [CD45.1] cells) were transferred 2 days before intravenous immunization with antigenic peptides in the absence (DMSO) or presence of TLR4 agonists. The numbers of cells present in primary (spleen) and secondary (lung) organs 3.5 days after immunization were determined. (B) Numbers of antigen-specific CD4 (OTII) and CD8 (OTI) T cells recovered in each tissue were determined by flow cytometry. Results are expressed as fold increases, calculated as the number of cells recovered from adjuvant-treated mice divided by the average number of cells recovered from the group treated with the antigen only, with no adjuvant (DMSO). Data are from two independent experiments, with triplicate mice used in each. Results for the individual recipients from experiments 1 (circles) and 2 (triangles) are shown. The horizontal line indicates the average fold increase for the combined groups. Asterisks indicate significant differences from the DMSO control group as determined by one-way ANOVA and Newman-Keuls post hoc analysis (P < 0.05).

Fig 4

LT-LPS preparations have lower relative effects than SmLPS or SmMPLA through TLR4/MD2 on either the MyD88- or the TRIF-dependent branch. BM-DC were treated with increasing concentrations of the LPS preparations for 2 h. qRT-PCR was performed to detect fold increases in steady-state levels of the MyD88-dependent endothelin-1 and IL-10 transcripts or the TRIF-dependent IFIT1 and IP-10 transcripts. Results are expressed as means ± standard errors of the means for triplicate determinations from one of two independent experiments.

Fig 5

LT-LPS preparations activate primary human monocytes. Primary human monocytes, enriched from PBMC preparations by adherence to plastic, were treated with increasing concentrations of the LPS preparations for 2 h. qRT-PCR was performed to quantify relative increases in levels of the inflammatory markers IL-6 and endothelin-1 and the type I IFN-dependent marker IFIT1. Results are expressed as means ± standard errors of the means for triplicate determinations from one of two independent experiments.