Synthetic mycobacterial molecular patterns partially complete Freund’s adjuvant

Abstract

Complete Freund's adjuvant (CFA) has historically been one of the most useful tools of immunologists. Essentially comprised of dead mycobacteria and mineral oil, we asked ourselves what is special about the mycobacterial part of this adjuvant, and could it be recapitulated synthetically? Here, we demonstrate the essentiality of N-glycolylated peptidoglycan plus trehalose dimycolate (both unique in mycobacteria) for the complete adjuvant effect using knockouts and chemical complementation. A combination of synthetic N-glycolyl muramyl dipeptide and minimal trehalose dimycolate motif GlcC14C18 was able to upregulate dendritic cell effectors, plus induce experimental autoimmunity qualitatively similar but quantitatively milder compared to CFA. This research outlines how to substitute CFA with a consistent, molecularly-defined adjuvant which may inform the design of immunotherapeutic agents and vaccines benefitting from cell-mediated immunity. We also anticipate using synthetic microbe-associated molecular patterns (MAMps) to study mycobacterial immunity and immunopathogenesis. Infection with Mycobacterium tuberculosis, or administration of bacille Calmette-Guérin (BCG), normally leads to cell-meditated immunity (CMI) to the corresponding bacterial antigens. The tuberculin skin test is positive when the immune response, a type IV hypersensitivity reaction or delayed-type hypersensitivity (DTH), occurs to tuberculin (a protein extract of M. tuberculosis). This "cutaneous sensitivity" was closely examined in the 1940s using complete Freund's adjuvant (CFA, heat-killed M. tuberculosis in mineral oil plus surfactant) 1,2. These studies provided the first direct evidence for the cellular nature of DTH by transfer from a CFA-immunized guinea pig to a naïve one only through the washed, heat-liable cellular fraction of peritoneal exudates 1,2. It is now known that DTH is mediated specifically by antigen-sensitive T cells. Today, CFA is a 'gold standard' adjuvant for eliciting CMI in research models of autoimmune disease. Notable is the experimental autoimmune encephalomyelitis (EAE) model of T-cell meditated destruction of myelin causing ascending paralysis, used most often to model multiple sclerosis 3,4. CFA is not used in humans because of high reactogenicity 5. We have asked ourselves: what was the impetus for Jules Freund to develop his eponymous adjuvant with M. tuberculosis? For those conducting TB research, it is well appreciated that handling M. tuberculosis (a slow growing, clumping, fastidious, and lethally pathogenic organism) is a significant task per se. So, why did Freund choose to incorporate these bacteria? Effort has been made to describe the microbe-associated molecular patterns (MAMPs) in mycobacteria that drive the adjuvant effect. Evidence has pointed to mycobacterial peptidoglycan (PGN), specifically down to the molecular motif muramyl dipeptide (MDP) 6,7. Mycobacteria are distinct in that they produce the N-glycolyl MDP motif in their cell wall 8-10 and this PGN modification has been shown to be more ...

Underwhelming or Misunderstood? Genetic Variability of Pattern Recognition Receptors in Immune Responses and Resistance to Mycobacterium tuberculosis

“…Mycobacteria produce a distinct NOD2 ligand, N-glycolyl MDP, while most other bacteria produce N-acetyl MDP (84,85). N-glycolylated peptidoglycan and MDP were shown to be better inducers of immune responses compared to the N-acetylated forms by comparing with mycobacterial KOs and synthetic MDPs (86)(87)(88). The absence of NOD2 during Mtb infection was accompanied by reduced nitric oxide and cytokine production from mouse macrophages (89,90) and reduced iNOS from human macrophages (91).…”

“…NOD2 signalling has been called “non-redundant” in that although there are shared pathways with TLR and CLR signalling (e.g. NF-κB), NOD2 signalling appears to work synergistically with other MAMPs and little immune response is produced with MDP stimulation alone ( 86 , 88 , 92 ).…”

Identification of CTL Epitopes on Efflux Pumps of the ATP-Binding Cassette and the Major Facilitator Superfamily of Mycobacterium tuberculosis

“…Analysis of the in vitro LDH lysis activity of these three peptides ( Figure 3(c) ) showed that Rv1218c-p24 and the Rv2477c-p182-induced T cells lysed peptide-loaded T2A2 cells, so these two peptides were selected for an in vivo assay. Several studies have shown that complete Freund's adjuvant (CFA) significantly enhances the phagocytosis by macrophage in mice, and that this is closely associated with the improved immune response conferred by adjuvants [ 42 , 43 ]. In an in vivo experiment in mice, the CFA adjuvant was subcutaneously injected first into the mouse, then the antigenic peptide was subcutaneously emulsified with IFA.…”

Serjania marginata Casar. hydroalcoholic extract reduced cytokine and inflammatory parameters in experimental models of inflammation and infection in mice

“…The EESM was also tested against CFA (an oil suspension containing killed M. tuberculosis), an immunogenic/in ammatory stimulus since it is not possible to work with viable M. tuberculosis (Dubé et al, 2020). The CFA induced persistent edema, mechanical hyperalgesia and cold sensitivity in control animals for 22 days.…”