Modulation of adaptive immunity by different adjuvant-antigen combinations in mice lacking Nod2

Abstract

The mechanisms underlying adjuvant effects are under renewed scrutiny because of the enormous implications for vaccine development. Additionally, new low-toxicity adjuvants are sought to enhance vaccine formulations. Muramyl dipeptide (MDP) is a component of the peptidoglycan polymer and was shown to be an active but low-toxicity component of complete Freund's adjuvant, a powerful adjuvant composed of mycobacteria lysates in an oil emulsion. MDP activates cells primarily via the cytosolic NLR family member Nod2 and is therefore linked to the ability of adjuvants to enhance antibody production. Accordingly, we tested the adjuvant properties of the MDP-Nod2 pathway. We found that MDP, compared to the TLR agonist lipopolysaccharide, has minimal adjuvant properties for antibody production under a variety of immunization conditions. We also observed that the oil emulsion incomplete Freund's adjuvant (IFA) supplanted the requirements for the TLR pathway independent of the antigen. Surprisingly, we observed that Nod2 was required for an optimal IgG1 and IgG2c response in the absence of exogenous TLR or NLR agonists. Collectively, our results argue that oil emulsions deserve greater attention for their immunostimulatory properties.

Fig. 1

Antibody production by C57BL/6 mice immunized with HSA in the presence of MDP or LPS. Mice were primed at day 0 and boosted at days 14 and 21. At 42 days, mice were bled and sacrificed. All serum time points were processed at the same time to determine the HSA-specific titers for each isotype. Each point represents the serum antibody titer to HSA for an individual mouse and titer values represent reciprocal serum dilution yielding half-maximal signal. *, p<0.05.

Fig. 2

Antibody production by C57BL/6 and Nod2-deficient mice immunized with HSA in the presence or in the absence of MDP or LPS. Mice were immunized as described in Fig. 1 and serum anti-HSA titers determined by ELISA. *, p<0.05.

Fig. 3

Antibody production by C57BL/6 mice after peritoneal injection with TB-OTII-NE236 peptide plus IFA in the presence or in the absent of MDP or LPS. Mice were immunized at day 0 and boosted at days 14 and 21 with TB-OTII-NE236 in IFA alone, IFA and LPS or IFA and MDP. Each point represents the serum antibody titer to NE236 for an individual mouse and titer values represent reciprocal serum dilution yielding half-maximal signal.

Fig. 4

Antibody production by C57BL/6 or Nod2-deficient mice immunized with TB-OTII-NE236 peptide plus IFA (top graphs) or IFA+LPS (lower graphs). Mice were immunized as described in Fig. 1 and serum titers to NE236 measured by isotype-specific ELISA. *, p<0.05.

Fig. 5

Immunization of control (Myd88^+/+) or MyD88-deficient mice immunized with TB-OTII-NE236 peptide plus IFA. Mice were immunized as described in Figs 1. Shown are IgG1 antibody titers specific for NE236. Antigen-specific IgM, IgG2b and IgG2c responses were also determined in these experiments and did not show any significant differences comparing control or Myd88^−/− mice.