TLR4-based adjuvant - MPLA-SM VacciGrade™

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

MPLA-SM* VacciGrade is a pre-clinical preparation of Monophosphoryl Lipid A (MPLA), a potent TLR4 agonist and vaccine adjuvant.

MPLA-SM is extracted from the lipopolysaccharide (LPS) of Salmonella minnesota Re595 (Re mutant), a rough strain of Gram-negative bacteria, using acid and heat treatment, followed by chromatography [1]. While LPS toxicty prohibits its clinical use, its MPLA subunit is used for vaccine adjuvantation [2]. 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 [3-5]. 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 [4].

 More details

MPLA-SM* VacciGrade™, like our previous reference MPLA-SM VacciGrade™ (removed from our catalog), contains a mix of MPLA congeneric forms differing in the number of acyl chains. It has been suggested that this congener mix is responsible for the partial TLR4 agonist function of some preparations [7]. MPLA-SM* VacciGrade™ 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). 

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

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

Key features:

• Agonist of mouse and human TLR4
• Strong inducer of Th1 immune response
• Negligible TLR2 activity
• Each lot is functionally tested
   

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

References:

1. 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.
2. Del Giudice G. et al., 2018. Correlates fo adjuvanticity: a review on adjuvants in licensed vaccines. Semin. Immunol. 39:14-21.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.