TLR4-based adjuvant - MPLA-SM VacciGrade™

Monophosphoryl Lipid A from Salmonella minnesota R595 - TLR4-based adjuvant

InvivoGen provides two preparations of Monophosphoryl Lipid A (MPLA) extracted from the lipopolysaccharide (LPS) of Salmonella minnesota Re595 (Re mutant), a rough strain of Gram-negative bacteria, for Toll-like receptor 4 (TLR4) activation. 

While the toxicity associated with LPS prohibits its potential clinical use, MPLA is used for vaccine adjuvantation  [1]. Indeed, MPLA is considerably less toxic than LPS whilst maintaining immunostimulatory activity. MPLA has been tested as an adjuvant in mice and reported to induce a strong Th1 response [2-4]. Although the mechanism of action of MPLA has not been fully elucidated, it has been suggested that MPLA improves vaccine immunogenicity by enhancing antigen-presenting cell maturation [3].

MPLA-SM* and MPLA-SM are extracted from LPS using treatment with acid and heat followed by chromatography [5].  These preparations contain a mix of MPLA congeneric forms differing in the number of acyl chains. It has been suggested that this mix is responsible for the partial TLR4 agonist function of some preparations [6]. MPLA-SM* is a new reference in our catalog. It results from an improved process of MPLA-SM extraction. While MPLA-SM* and MPLA-SM have the same ability to activate murine TLR4, MPLA-SM* is more potent than MPLA-SM at inducing human TLR4 responses (see Figures). 

 More details

VacciGrade™ is a high-quality pre-clinical grade.

MPLA-SM* VacciGrade™​ is   for research use only, and not for human or veterinary

Key features:

• Agonists of mouse and human TLR4
• Strong Th1 responses inducers
• Negligible TLR2 activity
• Each lot is functionally tested
   

Note: MPLA-SM* Vaccigrade™ is also available in a standard grade as MPLA-SM*.

References:

1. Del Giudice G. et al., 2018. Correlates fo adjuvanticity: a review on adjuvants in licensed vaccines. Semin. Immunol. 39:14-21.
2. Fransen F. et al., 2007. Agonists of Toll-like receptors 3, 4, 7, and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup. Infect Immun. 75(12) :5939-46.
3. Rhee EG. et al., 2010. TLR4 Ligands Augment Antigen-Specific CD8+ T Lymphocyte Responses Elicited by a Viral Vaccine Vector. J. Virol. 84: 10413 - 10419.
4. Didierlaurent A. et al., 2009. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity. J Immunol 183(10): 6186-97.
5. Qureshi N. et al., 1985. Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high-performance liquid chromatography and complete structural determination. J. Biol. Chem. 260, 5271–8.
6. Wang YQ. et al., 2020. MPL Adjuvant Contains Competitive Antagonists of Human TLR4. Front. Immunol. 11:577823.

Figures

MPLA-SM and MPLA-SM* induce a similar dose-dependent response in HEK-Blue™ mTLR4 cells. The cells were incubated with increasing concentrations of two preparations of S. minnesota monophosphoryl lipid A, MPLA-SM, and MPLA-SM*. After overnight incubation in HEK-Blue™ detection medium, a SEAP detection growth medium, the activation of mouse (m)TLR4 was assessed by determining the presence of SEAP in the supernatant. Data are expressed as optical density at 630 nm (±SEM).

MPLA-SM* is more potent than MPLA-SM at inducing a dose-dependent response in HEK-Blue™ hTLR4 cells. The cells were incubated with increasing concentrations of two preparations of S. minnesota monophosphoryl lipid A, MPLA-SM and MPLA-SM*. After overnight incubation in HEK-Blue™ detection medium, a SEAP detection growth medium, the activation of human (h)TLR4 was assessed by determining the presence of SEAP in the supernatant. Data are expressed as optical density at 630 nm (±SEM).

Specifications

Species: Salmonella enterica serovar minnesota mutant R595

Description: TLR4 agonist VacciGrade™

Polarization of adaptive immune response: Th1 response

Working concentration: 2- 20 μg/mouse

Appearance: Clear lipidic film

Solubility: 1 mg/ml in DMSO

Quality control:

• Sterility guaranteed
• Biological activity has been tested using HEK-Blue™ hTLR4 cells
• The presence of other bacterial components (e.g. lipoproteins) is controlled using HEK-Blue™ TLR2 cells

Contents

MPLA-SM* VacciGrade™ is provided as a clear, lyophilized lipidic film.

• 1 mg of lyophilized MPLA-SM* VacciGrade™
• 10 ml sterile endotoxin-free physiological water (NaCl 0.9%)

 The product is shipped at room temperature.

 Store at -20°C. Upon resuspension, prepare aliquots and store them at -20°C. 

 The product is stable for 1 year when properly stored upon receipt, and for 6 months when properly stored upon resuspension.

 Avoid repeated freeze-thaw cycles.

VacciGrade™

VacciGrade™ is a high-quality pre-clinical grade. VacciGrade™ products are filter-sterilized (0.2 µm) and filled under strict aseptic conditions in a clean room*. The absence of bacterial contamination is assessed by a sterility test using a pharmacopeia-derived assay. The level of bacterial contaminants (endotoxins and lipoproteins) in each lot is verified using a LAL assay and/or a TLR2 and TLR4 reporter assay.
*Except for LPS VacciGrade™, which is prepared in a laminar flow hood dedicated to LPS.

Details

Monophosphoryl Lipid A (MPLA) is a natural compound extracted from the LPS component of the cell wall of Gram-negative bacteria. LPS is a potent activator of the immune system. Its recognition by TLR4 leads to NF-κB and IRF activation and the production of proinflammatory cytokines and interferons, respectively [1]. Thus, LPS features many characteristics needed for an effective vaccine adjuvant. However, large uncontrolled amounts of LPS are extremely toxic and can cause devastating diseases [2].

Wild-type LPS, referred to as smooth (sLPS) comprises three covalently linked regions: a Lipid A backbone, an oligosaccharide core, and O-polysaccharide chains. Some bacteria produce a truncated LPS, without O-side chains, referred to as rough (rLPS) [3]. LPS biological activity is mediated by Lipid A recognition by TLR4 and is commensurate to Lipid A number of fatty acyl chains [2]. Hexa-acylated (6 chains) Lipid A is a highly potent TLR4 agonist, while under‑acylated (4-5 chains) Lipid A induces lower or antagonistic responses [4].

Acidic extraction of Lipid A from LPS produces monophosphoryl Lipid A (MPLA), which displays reduced toxicity while retaining the ability to activate TLR4 [5,6]. The reduced toxicity of MPLA is attributed to the preferential triggering of the IRF pathway upon TLR4 activation, resulting in decreased induction of inflammatory cytokines [7].

References:

1. Kuzmich, NN. et al., 2017. TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis. Vaccines (Basel) 5unol. 165(2):618-22.
2. Steimle, A. et al., 2016. Structure and function: Lipid A modifications in commensals and pathogens. Int J Med Microbiol 306, 290-301.
3. Raetz CR. 1990. Biochemistry of endotoxins. Annu. Rev. Biochem. 59, 129‑70.
4. Cochet, F. & Peri, F. 2017. The role of carbohydrates in the lipopolysaccharide (LPS)/Toll-Like Receptor 4 (TLR4) Signalling. Int J Mol Sci 18.
5. Qureshi N. et al., 1982. Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium. J. Biol. Chem., 257:11808-15.
6. Romero CD. et al., 2011. The Toll-Like Receptor 4 agonist monophosphoryl Lipid A augments innate host resistance to systemic bacterial infection. Infect Immun. 79: 3576–3587.
7. Mata-Haro V. et al., 2007. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science. 316(5831):1628-32.