Biosynthetically engineered lipopolysaccharide as vaccine adjuvant.

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Biosynthetically engineered lipopolysaccharide as vaccine adjuvant Expert Rev. Vaccines Early online, 1–16 (2015)

Afshin Zariri1,2 and Peter van der Ley*1 1 Institute for Translational Vaccinology (InTraVacc), Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, The Netherlands 2 Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands *Author for correspondence: Tel.: +31 030 274 2533 [email protected]

Lipopolysaccharide (LPS), a dominant component of the Gram-negative bacterial outer membrane, is a strong activator of the innate immune system, and thereby an important determinant in the adaptive immune response following bacterial infection. This adjuvant activity can be harnessed following immunization with bacteria-derived vaccines that naturally contain LPS, and when LPS or molecules derived from it are added to purified vaccine antigens. However, the downside of the strong biological activity of LPS is its ability to contribute to vaccine reactogenicity. Modification of the LPS structure allows triggering of a proper immune response needed in a vaccine against a particular pathogen while at the same time lowering its toxicity. Extensive modifications to the basic structure are possible by using our current knowledge of bacterial genes involved in LPS biosynthesis and modification. This review focuses on biosynthetic engineering of the structure of LPS and implications of these modifications for generation of safe adjuvants. KEYWORDS: adjuvant . lipid A . lipopolysaccharide . oligosaccharide . outer membrane vesicle . pathogenic bacteria .

TLR4

.

vaccine

Due to increasing safety demands, side effects concerns, increasing knowledge and implementation of new techniques, development of vaccines has shifted from inactivated whole organisms toward the use of highly purified recombinant proteins and single antigens. However, the reliance on vaccines with a more restricted, better-defined composition has necessitated the inclusion of adjuvants. Adjuvants are defined as molecules that provide ‘help’ to an antigen. This ‘help’ can result in an alteration of the quality and magnitude of immune responses against vaccine antigens. Adjuvants can improve immune responses in poorly responsive populations, broaden cross protection, induce a shift toward a particular T helper response and reduce the amount of antigen needed in a vaccine [1]. The mammalian immune system recognizes the presence of pathogens through pattern recognition receptors capable of detecting conserved parts of pathogens that are important for their survival. These pathogen-associated molecular patterns (PAMPs) include DNA and RNA from microorganisms (bacteria and viruses), protein or lipid components, and receptor

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10.1586/14760584.2015.1026808

binding of PAMPs triggers downstream signal transduction pathways, leading to activation of the innate immune system [2,3]. Therefore, various molecules derived from microbes can be used as adjuvants through their potential to interact with mammalian immune cells. Lipopolysaccharide (LPS), a part of the outer membrane (OM) of gram-negative bacteria, generally constitutes 35–45% of the OM. The rest of the OM typically consists of 30–40% protein and 25% lipids [4]. LPS contributes greatly to the structural integrity of bacterial cells and is essential for the barrier function of the OM against harmful chemicals. LPS typically consists of three parts, a polysaccharide O-antigen, a core-oligosaccharide and a hydrophobic lipid A. The lipid A is recognized by mammalian immune cells through the pattern recognition receptor complex toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2) resulting in activation of immune cells and the release of inflammatory cytokines. LPS is one of the most active PAMPs known, and is already active in the picogram range. LPS is a potent inducer of innate immunity and a potent enhancer of the immune response

2015 Informa UK Ltd

ISSN 1476-0584

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Review

Zariri & van der Ley

E. coli

N. meningitidis

O OH P O OH O O O O 14


14

O

O 12

O O

B. pertussis

OH

OH

NH

O

O HO 14

O

HO O

14

O NH O P OH O HO OH 14

O OH P O OH O O HO 12

OH

O

O O

O

O

NH

HO O HO

14 12

12

O O O 12

O O

NH

O O P OH OH

14

O OH P O OH O O HO

O O O

14

O

O

NH

HO

14 14

O HO 10

O O O HO

NH

O O P OH OH

14

Figure 1. Typical wild-type lipid A structures of Escherichia coli, Neisseria meningitidis and Bordetella pertussis. The numbers show the fatty acid chain length.

against protein antigens. As an adjuvant, LPS is also capable of producing a shift in the T cell response toward Th1 [5]. However, the use of LPS as an adjuvant is severely limited by its endotoxic activity, leading to unacceptable reactogenicity. Therefore, efforts have been made to attenuate LPS and to separate endotoxic and immunostimulatory activities, either by direct chemical treatment of purified LPS or by genetic modification of LPS biosynthesis. The purpose of this review is to describe the role of LPS in vaccines and give an overview of LPS modifications in the search for potential safe adjuvants. There are three ways to create modified LPS: by chemical treatment leading to partial degradation, by synthetic assembly or by genetic modification of the bacteria [6–9]. Some benefits of the latter approach are production of complete LPS molecules as opposed to only the lipid A or oligosaccharide moieties, the ease to produce a variety of structures just by growing the bacteria, the lack of need to remove chemical or enzymatic additives and the possibility to modify LPS as an intrinsic component of live, whole cell or OM vesicle (OMV) vaccines [10–14]. Here we will focus on the genetic modifications of LPS, as chemical modifications for creating lipid A derivatives such as monophosphoryl lipid A (MPLA) have already been discussed extensively in other reviews [15,16]. LPS structure, function & activity

The OM of gram-negative bacteria is an asymmetric bilayer composed of glycerophospholipids on the inner leaflet and predominantly LPS on the outer leaflet. Already in the 1960s, it was proposed that LPS of gram-negative bacteria has a common general architecture composed of three components: lipid A, core-oligosaccharide and O-specific side chain [17,18]. Later investigations with the use of mass spectrometry and high-field nuclear magnetic resonance spectroscopy revealed a more detailed structure of LPS. Lipid A is the hydrophobic part of LPS that is anchored in the OM. Its basic structure is a glucosamine disaccharide carrying four to six acyl chains and two phosphate groups (FIGURE 1) [18]. The core oligosaccharide part is covalently attached to the lipid A and can be divided into an inner and outer core. The inner core is closest to lipid A and consists of sugars such as 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno heptose, while the outer doi: 10.1586/14760584.2015.1026808

core typically consists of hexoses and hexosamines. In most cases, a polymer of repeating saccharide subunits named O-polysaccharide or O-chain is attached to the outer core. In several gram-negative bacteria, for example Neisseria meningitidis, which colonizes mucosal surfaces, this O-chain is lacking and the resulting structure is often referred to as lipooligosaccharide instead of LPS [19]. A potential outcome of gram-negative infection is septic shock, of which many symptoms can be attributed to the LPS, more precisely the lipid A part of LPS. The long-sought receptor by which LPS triggers the innate immune system was identified by the genetic investigation of a mouse strain that failed to respond to LPS. It turned out to be a member of a family of transmembrane pattern recognition TLRs which recognize diverse microbial components, also known as PAMPs, with TLR4 forming the LPS receptor in complex with MD-2 [20,21]. There are 10 known human TLRs and each recognizes particular classes of PAMPs such as bacterial lipoproteins, bacterial DNA, viral RNA or bacterial flagella, among others. TLR1, 2, 4, 5 and 6 are expressed on the cell surface, while TLR3, 7, 8 and 9 are found in endosomal compartments, although upon ligand recognition, TLR4 also can migrate to and signal from the endosomal compartment [2]. Upon recognition of a ligand, TLRs form homo- or heterodimers, thereby launching a signaling cascade. Typically, this signaling cascade starts with the recruitment of adaptor molecules such as myeloid differentiation primary response gene 88 (MyD88), Toll/IL-1 receptor (TIR)-domain-containing adapter-inducing IFN-b (TRIF), TIR-containing adaptor protein and TRIF-related adaptor molecule [2,22]. All TLRs, except for TLR3, signal via MyD88. In the case of TLR1, TLR2 and TLR6, signaling through MyD88 occurs with addition of TIR-containing adaptor protein. TLR3 signals solely via TRIF, whereas TLR4 uses TIRcontaining adaptor protein to bind MyD88 at the cell surface and TRIF-related adaptor molecule to bind TRIF in the endosomal compartment. Thus, TLR signaling is broadly characterized as MyD88 dependent or independent (TRIF dependent). MyD88-dependent signaling recruits the IL-1 receptorassociated kinase family members, which, in turn, activate the kinases TNF-associated factor 6 (TRAF 6), TGF-b–activated kinase 1 and the IkB kinase (IKK) complex, leading to the

Expert Rev. Vaccines

Biosynthetically engineered LPS as vaccine adjuvant


translocation of NF-kB to the nucleus and the transcription of pro-inflammatory cytokines [2,22]. TRIF-dependent signaling activates the TRAF 3 leading to activation of TRAF family member-associated NFkB activator binding kinase 1 and IKKi. TRAF family member-associated NFkB activator binding kinase 1 and IKKi subsequently activate TRAF family memberassociated NFkB activator which phosphorylates interferon regulatory factor 3 and interferon regulatory factor 7, ultimately resulting in the production of type I interferons [2,3,22]. Lipid A biosynthesis

Lipid A, the hydrophobic part of LPS, is responsible for most of its activation of the innate immune response. The TLR complex TLR4/MD-2 recognizes lipid A, and subsequent dimerization of TLR4 results in signaling through adaptor molecules MyD88 as well as TRIF. Ultimately, signaling through the adaptor molecules and translocation of NF-kB starts the secretion of inflammatory cytokines such as IL-6 and TNF-a. The biosynthesis of lipid A has been investigated most extensively in Escherichia coli, but the nine enzymes described by Raetz and Whitfield as required for the biosynthesis of Kdo2lipid A and their encoding genes are conserved among practically all gram-negative bacteria (TABLE 1) [6]. Even in the plant Arabidopsis thaliana, the lipid A biosynthesis pathway was discovered, and it shares great resemblance to the lipid A biosynthesis pathway of E. coli [23]. The first precursor molecule needed for lipid A formation is UDP-N-acetylglucosamine (GlcNAc), and the first step in the pathway, fatty acylation of UDP-GlcNAc, is catalyzed by LpxA. Subsequently, LpxC deacylates UDP-3-O-(acyl)-GlcNAc in the first committed step of lipid A biosynthesis. The next step is addition of a second R-3-hydroxymyristoyl chain by LpxD to form UDP-2,3-diacylGlcN. LpxH cleaves the pyrophosphate linkage of UDP-2,3diacyl-GlcN to form 2,3-diacyl-GlcN-1-phosphate (lipid X), and the 1¢-6–linked disaccharide characteristic of all lipid A molecules is then created by LpxB which condenses lipid X with UDP-2,3-diacyl-GlcN and releases UDP. LpxK, KdtA, LpxL and LpxM catalyze the final four steps of the Kdo2-lipid A synthesis pathway. LpxK phosphorylates the 4¢-position of the disaccharide 1-phosphate to form lipid IVa, the minimal LPS structure necessary for viability of E. coli. KdtA typically adds at least two Kdo residues, although in some species like Haemophilus influenzae and Bordetella pertussis, it incorporates only one Kdo residue [24,25]. The final steps of Kdo2-lipid A biosynthesis include addition of the secondary lauroyl and myristoyl residues to the primary 3-OH fatty acid chains at 2¢ and 3¢ positions of lipid A by LpxL and LpxM, respectively [6,9]. Some biosynthetic steps in the lipid A pathway can be turned off, resulting in bacterial strains with a modified lipid A structure. For example, in most bacteria, it is possible to inactivate the LpxL and LpxM homologs, leading to loss of one or both secondary acyl chains [26]. However, most of the early steps seem to be essential for bacterial survival, therefore making a knockout of those genes not feasible. Whether it is possible to remove an enzyme involved in the biosynthesis of lipid informahealthcare.com

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A is often dependent on the bacterial species. In contrast to E. coli, a knockout mutation in lpxA, the gene required for the first step in the synthesis of lipid A, is possible in N. meningitidis, providing the first example of a gram-negative species completely deficient of LPS [27]. Similarly, a knockout of lpxH, responsible for cleavage of the pyrophosphate linkage of UDP-2,3-diacyl-GlcN, is detrimental to the survival of E. coli but possible in N. meningitidis [28]. There have been two other instances published of gram-negative bacterial species which can survive with complete loss of LPS, Moraxella catarrhalis and Acinetobacter baumannii [29,30]. Only for A. baumannii and N. meningitidis, naturally occurring LPS-deficient mutants have been described where the bacteria had spontaneously turned off LPS biosynthesis by mutation of their lpxD or lpxH gene, respectively [29,31]. Overall, a diphosphate hexa-acylated lipid A structure is the most potent activator of TLR4/MD-2. Alterations made to the lipid A structure rendering a penta-acyl or hepta-acyl lipid A typically have reduced human TLR4/MD-2 activating ability and, in most cases, tetra-acylated lipid A is either an antagonist or induces very little activity which is seen in the case of lipid IVa from E. coli [32,33]. The loss of phosphate groups at the 1 and 4 positions typically induces a reduced TLR4/ MD-2 activation due to interactions of the phosphate groups during dimerization of TLR4/MD-2 [32]. Alteration of lipid A has great influence on the dimerization and downstream signaling of TLR4/MD-2. Moreover, recognition of lipid A by the TLR4/MD-2 complex is species-specific. For instance, lipid IVa is considered an antagonist of human TLR4, but an agonist of murine TLR4. Crystal structure studies have revealed that this difference of activity starts with altered fitting of the lipid IVa in the hydrophobic pocket of MD-2 [34]. In murine MD-2, the negatively charged glutamate residue (E122) deflects the negatively charged phosphate group, thereby pushing the backbone upward. This causes the lipid IVa to sit higher and one of the fatty acid chains of the lipid IVa to be partially surface exposed similar to hexa-acylated lipid A [34,35]. This positioning makes it possible for the phosphate groups present in the lipid A and the fatty acid chain outside the pocket to interact with parts of TLR4 and MD-2 during dimerization leading to downstream signaling and activity. In human TLR4, lipid IVa is not pushed upward and all four fatty acid chains of lipid IVa are positioned deep inside the hydrophobic pocket of MD-2 causing a loss of interaction of the phosphate groups and fatty acid chain during dimerization [34,35]. Species specificity of TLR4/MD-2 poses a problem for the use of murine animal models when testing altered LPS structures that are meant for use as adjuvants in humans. Therefore, more insight into LPS and species-specific TLR4/MD-2 interactions is highly important for good predictability of the effects of modified LPS adjuvants. Immune modulation during infection: evidence from mutants

Modification of LPS is a strategy used by many gram-negative pathogenic bacteria to evade immune recognition and killing doi: 10.1586/14760584.2015.1026808

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Table 1. Biosynthesis enzymes for the formation of Kdo2-lipid A: the Raetz pathway. Structure OH O

HO O O

O O

NH

O


O O

O

NH 2

O

O O

O O O

NH

O O

Lipid X

OH

O

O

HO O O O

NH

O

O O

O-UDP

NH O O

O O

O O O

O

O

NH

HO

HO HO

O O

OH

O HO P O OH O O O

O O

O

NH

O O

O

O

NH

HO O O

HO O HO OH

HO

O O

NH

LpxH

Cleaves the pyrophosphate linkage of UDP-2,3-diacyl-GlcN to make lipid X

LpxB

Condenses UDP-2,3-diacyl-GlcN with lipid X to form the 1¢-6-linkage in lipid A

LpxK

Phosphorylates the 4¢-position of the disaccharide 1-phosphate to form lipid IVa

KdtA

Incorporates typically two Kdo residues to the 6¢-position of lipid IVa

LpxL

Adds a secondary lauroyl residue to the fatty acid chain at 2¢-position of lipid A

LpxM

Adds a secondary lauroyl residue to the fatty acid chain at 3¢-position of lipid A

O O P OH OH

OH O O OH O

O O

O

O

NH

HO O O

O O O O

NH

O O P OH OH

OH HO O HO OH

HO

Adds a second R-3-hydroxymyristate chain to make UDP-2,3-diacyl-GlcN

O O P OH OH

O

O HO P O OH O O O

O

LpxD

O

O

HO HO

Catalyzes the deacetylation of UDP-3-O(acyl)-GlcNAc Is the committed step of lipid A biosynthesis

O

OH O

LpxC

O O P OH OH

OH HO

Catalyzes the fatty acylation of UDP-GlcNAc Requires thioester R-3-hydroxymyristoyl acyl carrier protein

O-UDP

OH HO

LpxA

O-UDP

OH HO

Function

O-UDP

OH HO

Enzyme

OH O O OH

O O HO P O OH O O O O

O

O

O O

14 14

O

O

NH

HO O O

O O O O

14 12

NH

O O P OH OH

14 14

Each subsequent addition in the lipid A-Kdo2 structure is indicated in red. The glucosamine backbone is black and the Kdo disaccharide is in green. GlcNAc: N-acetylglucosamine; Kdo: 3-deoxy-D-manno-octulosonic acid.

doi: 10.1586/14760584.2015.1026808



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Table 2. Lipid A modifying enzymes. Structure

O OH P O OH O O O O O 14

O O

O

O

NH

HO O O

O O O O

14 12

14

NH

O O P OH OH

14

Function

LpxE

Removes 1-phosphate group

LpxF

Removes 4¢-phosphate group

LpxR

Removes acyl chain(s) from 3¢-position

PagL

Removes acyl chain from 3-position

LmtA

Catalyzes methylation of 1-phosphate

LpxO

Adds hydroxyl group to fatty acid myristate at 3¢-postion

PagP

Adds palmitate to primary linked acyl chain at 2-position

PmrC

Adds phosphoethanolamine at 1-position

14

O

LpxF

Modified lipid A


Unmodified lipid A

OH

Enzyme

ArnT H2N HO

OH

OH

O O P O OH O O O O HO

LpxO

O

14

O

NH

HO O O

O

LpxE O

14 12

14

O O

O

O

O O

NH

PmrC NH3

14 14

LpxR

O O O P O P O OH OH

PagL PagP

O O P O P OH LpxT OH OH O P O OH

ArnT

Adds aminoarabinose at 4¢-position

LpxT

Adds phosphate group at 1-phosphate

CH3 LmtA

Colored parts indicate the specificities of the different modifying enzymes. The numbers show the fatty acid chain length.

by immune cells, thereby enhancing their survival in a host. Modification of the lipid A moiety to evade immune recognition can be achieved by changing the charge of the bacterial surface through the addition of phosphoethanolamine or aminoarabinose residues, which increases resistance to innate immune effectors, or by changing the structure of lipid A in such a way that recognition by the TLR4/MD-2 receptor complex is directly affected [36]. The particular lipid A acylation and phosphorylation pattern is crucial for its recognition; therefore, removal of acyl or phosphate groups by gram-negative pathogens is a common immune evasion strategy (TABLE 2). Salmonella enterica serovar Typhimurium has a basic unmodified lipid A structure which is identical to E. coli lipid A. However, for survival in host cells where environmental stressors such as cationic antimicrobial peptides (CAMPs) and low pH are present, this pathogen can utilize a range of LPS modification enzymes. These include EptA and ArnT for the addition of phosphoethanolamine and aminoarabinose, respectively, PagP and PagL for adding and removing a single acyl chain, respectively, and LpxR for removal of the 3¢-acyloxyacyl group. EptA or ArnT is induced by exposure of cells to a mild acidic environment [9]. The aminoarabinose group added by ArnT neutralizes the negative charge of the lipid A 4¢-phosphate group, making it less susceptible to CAMPs and polymyxin. Palmitate added by PagP is induced by low Mg2+ or CAMPs [9]. Deletion of LpxR in Salmonella typhimurium reduces the capability for intracellular growth of this pathogen in macrophages [37]. Some pathogens such as Yersinia pestis can survive in different host species and modify their lipid A structure accordingly. It expresses a hexa-acylated lipid A form inside the flea at temperatures between 21 and 27 C, while inside mammals at informahealthcare.com

37 C, the same pathogen expresses a tetra-acylated lipid A form that is a TLR4 antagonist [38]. Y. pestis strains engineered to make hexa-acylated lipid A at 37 C are avirulent in mice, showing the importance of TLR4 evasion through adaptation of lipid A biosynthesis [38]. Environmental factors can directly influence the lipid A structure of bacterial pathogens. Cystic fibrosis patients often develop chronic infection of the airways by Pseudomonas aeruginosa. Although P. aeruginosa can express a variety of different LPS structures, P. aeruginosa isolates from cystic fibrosis patients have a specific structure including addition of palmitate, aminoarabinose and removal of a 3-OH C10 fatty acid chain [39–41]. These palmitate and aminoarabinose additions are associated with resistance to CAMPs and increased inflammatory responses [41]. None of these LPS modifications have been observed in isolates from the environment or patients with other conditions [40]. Francisella tularensis and the related mouse pathogen Francisella novicida synthesize a lipid A structure lacking the 4¢-monophosphate group due to the activity of LpxF phosphatase. An LpxF deletion mutant of F. novicida which can no longer remove the 4¢-monophosphate group shows higher local cytokine induction, greater neutrophil influx and higher survival upon infection of mice. In addition, deletion of the LpxF phosphatase causes higher susceptibility to CAMPs such as polymyxin B. LpxF is, therefore, a virulence factor of Francisella, reducing the immune activation and host bactericidal activity [42,43]. Inactivation of lipid A biosynthesis genes is also used by some bacterial species to alter their lipid A structure, usually to evade host immunity. This was observed in N. meningitidis, doi: 10.1586/14760584.2015.1026808


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where approximately 10% of the clinical isolates were found to have LPS with penta-acylated lipid A due to lpxL1 mutations, and the mutant bacteria gave a strongly reduced cytokine induction in monocytes. Patients infected with an lpxL1 mutant strain had less severe systemic inflammation and reduced coagulopathy, showing that this mutation has an effect on the clinical course of meningococcal invasive disease [44]. In a mouse meningococcal infection model, the lpxL1 mutant displayed strongly reduced virulence, while it was increased in an lpxL2 mutant lacking the secondary C12 acyl chain at the 2 instead of 2¢position [45]. Gonococcal lpxL1 mutations have not been reported in patient isolates; but in a murine model of gonorrhea, they can still establish infection but show reduced localized inflammation [46]. Changes of the LPS structure can also influence barrier function of the OM. For instance, the naturally occurring meningococcal lpxL1 mutants showed an increased sensitivity to palmitic acid. Growth of these lpxL1 mutants was completely abolished on agar plates in the presence 150 mg/ml palmitic acid [47]. Reduced OM integrity may offset the advantage conferred by escape from innate immunity, and this could explain why lpxL1 mutation is not a universal feature in N. meningitidis. Mutation of the PmrC enzyme, which incorporates phosphoethanolamine in the lipid A of Citrobacter rodentium, caused enhanced susceptibility for antibiotics that have to diffuse across the LPS layer of the OM. In addition, PmrC mutant strains showed an increased influx of ethidium dye across the OM [48]. In N. meningitidis, the lptA enzyme adds a phosphoethanolamine group to the 1 and 4¢ positions of the lipid A, similar to PmrC in other bacterial species. Inactivation of lptA leads to reduced TNF-a induction in THP-1 cells, a human monocytic cell line, by meningococcal LPS or whole bacteria [49,50]. In contrast to the pathogenic Neisseria species and Neisseria lactamica, commensal Neisseria species lack the lptA gene and do not express phosphoethanolamine groups in their lipid A, and give a reduced inflammatory response [49]. The lptA deletion in Neisseria gonorrhoeae results in increased sensitivity to complement-mediated killing [51]. An lptA gonococcal mutant was attenuated during experimental human urethral infection, and it has been concluded that the phosphoethanolamine modification plays both a protective and immunostimulatory role [52]. Similarly, an arnT mutant of Bordetella bronchiseptica was not only more susceptible to polymyxin B, but also a higher amount of bacteria was needed to establish infection in mice, and in a transmission model, the arnT mutant had become inactive [53]. Although bacterial pathogens can use their abilities for LPS modification to enhance survival in a specific environment, these modifications can also result in reduced overall fitness. This was investigated in A. baumannii, where LpxA, LpxC, LpxD and PmrB mutants were tested for their fitness. The first three mutations result in complete loss of LPS, and mutation of PmrB leads to loss of phosphoethanolamine groups. While the mutant strains showed resistance to colistin, when they were compared to the wild-type strain, they had a reduced doi: 10.1586/14760584.2015.1026808

in vitro growth rate and a reduced fitness during in vitro and in vivo competitive growth [54]. LPS modifications are often used by gram-negative bacteria to circumvent detrimental effects of the environment toward the bacteria. Evidence from mutants carrying a modified LPS provides knowledge about the effect of these modifications and can potentially be harnessed for the development of novel LPS structures as vaccine adjuvants. Live vaccines based on lipid A mutants

There are two major advantages of using live vaccines, selfadjuvanting potential and the expression of the complete pathogen genome giving a wide selection of antigens. For the use of live microbes as a vaccine, attenuation of the strain is necessary to make it safe. The classical way of attenuating a strain is by repeated passages on a cell line or animal embryos. Live attenuated vaccines of F. tularensis and Y. pestis, the causative agents of tularemia and plague, respectively, have been developed in such a way. One of these live attenuated vaccine strains of F. tularensis developed by serial passaging is known as Live Vaccine Strain. Although it showed partial protection against the virulent strain, this tularemia vaccine was not licensed since there was a lack of understanding of attenuation, uncertain history and instability of the colony type [55]. In an alternative approach to attenuation, an F. novicida mutant lacking the lipid A 4¢-phosphatase LpxF has been investigated [43]. It was shown that the LpxF-mediated dephosphorylation of lipid A prevents a full activation of the innate immune response, and lpxF mutants may, therefore, be useful for immunization against tularemia. In Y. pestis, EV live attenuated vaccine strains have been made through serial passaging in which the most common characteristic is the loss of the pgm locus. The pgm locus encodes the pigmentation segment and high-pathogenicity island which carries the virulence genes needed for iron uptake [56]. EV strains have been used as human plague vaccines for decades. Furthermore, additional genetic modifications of the LPS have been utilized in pgm-deficient Y. pestis strains. Y. pestis contains a tetra-acyl lipid A structure in the human host at 37 C to circumvent triggering of the TLR4 pathway [38,57]. Acylation of the lipid A by insertion of the lpxL gene from E. coli permits the synthesis of a hexa-acylated LPS in Y. pestis at 37 C, a more potent agonist of TLR4. The LpxL-expressing attenuated Y. pestis strains retain the potential to prime T cells upon intranasal immunization in mice and can provide protection against challenge with the wild-type Y. pestis strain, while at the same time being avirulent [58,59]. This protection is mainly provided through T cells, because B cell depletion did not influence the vaccine efficacy [59]. Y. pestis strains grown at 22–28 C produce highly active hexa-acyl LPS due to the temperature-sensitive late acyltransferases, LpxM and LpxP. Hexa-acylated EV strains that are used for vaccinating humans often come with adverse effects, mainly caused by reactogenicity of their active hexa-acylated LPS. Mutation of the lpxM gene results in a penta-acyl LPS in Y. pestis EV strain grown at Expert Rev. Vaccines



22–28 C [60]. Subcutaneous inoculation with the DlpxM EV strain was harmless in pigs and Balb/c mice, whereas the parental strain showed noticeable toxicity in the latter [60]. In addition, the mutant strain induced increased immunity and provided better protection than the parental strain [60]. This increased protection was attributed to the pleiotropic effects of the lpxM mutation, which resulted in reduced expression and immunoreactivity of major surface proteins and carbohydrate antigens [61]. Live attenuated bacterial strains have also been used in vaccine development as a foreign antigen delivery system. Bacterial-based vaccines come with several advantages compared to other delivery systems, including low cost of production, the absence of animal products and genetic stability [62,63]. Listeria monocytogenes and Salmonella strains have been used most extensively as bacterial antigen delivery vehicles. L. monocytogenes and Salmonella are both intracellular pathogens, but they induce different immune responses due to residence in different subcellular locations. Antigens delivered by L. monocytogenes behave as endogenous antigens and are presented by MHC class I molecules resulting in strong cellmediated immune responses, whereas antigens delivered by Salmonella are presented in a MHC class II-mediated fashion inducing mainly Th2-type immune responses [62]. The major reason to use a pathogenic bacterial strain like Salmonella instead of a commensal is its capability to get to regional lymph nodes and the spleen after mucosal delivery, which is critical for the induction of long-lasting immunity. Rough mutants of Salmonella that lack O-antigen or part of the core are not potent as live oral vaccines (TABLE 3) [63,64]. Instead of direct deletion of genes involved in LPS synthesis, regulated expression of these genes provides possibilities to generate vaccine strains that synthesize LPS core and/or O-antigen at the moment of oral vaccination, but stop synthesis once the internal lymphoid tissues are reached, thereby retaining its function in colonization while reducing interference with the immune response against heterologous antigens [65]. Immunization of mice using recombinant attenuated Salmonella with regulated rfaH and carrying the pneumococcal protein PspA as heterologous antigen resulted in anti-PspA IgG antibody titers equal to or higher than the parent strain, whereas a complete knock out of rfaH lacking O-antigen gave significantly lower anti-PspA IgG titers [65]. Lysis of bacteria within the host will result in reactogenicity. A major inducer of this reactogenicity is LPS, so reducing its TLR4-activating capacity can also be applied to live vaccines. Expression of the lipid A modifying enzyme LpxE from F. tularensis in Salmonella selectively removes the 1-phosphate group of lipid A. This modification in combination with deletion of PagP, resulting in 4¢-monophosphoryl hexa-acylated lipid A, attenuated Salmonella approximately five orders of magnitude, while retaining its ability to transiently colonize the lymphoid tissues and its immunogenicity [12]. Other lipid A alterations investigated for reducing reactogenicity include mutation of msbB (also known as lpxM), which encodes an enzyme for adding a secondary myristic acid to lipid A. An informahealthcare.com

Review

msbB mutation in a tumor targeting Salmonella strain resulted in reduced TNF-a production and reduced virulence, while retaining similar tumor-targeting ability as the parent strain [66]. Although the msbB mutant showed reduced TNF-a production in vitro, an additional pagP mutation leading to loss of another secondary acyl chain was necessary to reduce the inflammatory responses also in vivo [67]. Thus, modification of lipid A provides a variety of approaches for expanding vaccine safety without reducing efficacy in live bacterial vaccines. Mutant LPS as intrinsic adjuvant

Whole cell vaccines are used against certain bacterial pathogens such as B. pertussis, the causative agent of whooping cough. Although whole cell vaccines of gram-negative bacteria usually give a good immune response, the presence of LPS and its associated endotoxic activity can lead to relatively high reactogenicity and ultimately unwanted side effects. As discussed above, many bacterial species harbor LPS-modifying enzymes that could reduce the toxicity of whole cell vaccines. Two such modifying enzymes, PagL and PagP, have been tested for improvement of B. pertussis whole cell vaccines. Upon expression, both enzymes resulted in an increase of IL-6 production induced by whole bacterial cells in the human monocytic cell line mono mac 6 (TABLE 3) [68]. PagP expression modified the LPS from penta-acylated to the highly active hexa-acylated form by addition of a fatty acid chain. However, PagL expression resulted in deacylation of LPS from a penta-acylated to a tetra-acylated form, which was expected to reduce its endotoxic activity. The reason for increase in activity of the PagL whole cell B. pertussis vaccine was attributed to increased release of LPS due to weakened LPS–LPS interaction in the membrane [68]. The whole cell vaccine expressing PagL was also tested in an immunization assay in mice, showing increased vaccine efficacy without alteration of the vaccine reactogenicity [11]. Another, more consistently effective approach to reduce B. pertussis LPS endotoxic activity is to block glucosamine modification of the lipid A phosphate groups by inactivating the arnT homolog [69,70]. When isolated LPS of an arnT-inactivated strain was compared to its isogenic wild-type strain in human macrophages, there was a clear reduction of IL-6 and TNF-a induction. This effect was not present with murine cells or cells expressing murine TLR4 [70]. Many studies have focused on the use of OMVs of gramnegative bacteria as a vaccine. OMVs are small spherical structures of 20–250 nm diameter, which are shed by bacteria. Biochemical comparison of OMVs and bacterial OM fractions has shown that OMVs mainly contain proteins and lipids normally present in the OM, with minor differences. These differences consist of some soluble periplasmic proteins that are retained in OMVs and some OM proteins such as porins that are enriched in OMVs. Several methods exist for the purification of OMVs. Naturally produced OMVs can be isolated by ultracentrifugation. Other methods use mechanical shearing, detergent extraction or chemical treatment. In addition, certain mutations can lead to increased OMV release [71,72]. doi: 10.1586/14760584.2015.1026808

Review


Table 3. Mutant lipopolysachharide as an intrinsic part of a vaccine. Bacterial strains

LPS modification

Lipid A structure

Mutation effect

Protection

Ref.

[27]


Live attenuated vaccines Francisella novicida

DlpxF

Less virulent in mice, stronger local cytokine response, greater influx of neutrophils, highly susceptible to polymyxin

Not tested

Yersinia pestis

lpxL

Avirulent in mice, T cell mediated protection

Decreased burden and increased survival against virulent Y. pestis

DlpxM

Avirulent in mice and quinea pigs

85–100% in outbred mice and guinea pigs

[44]

DwaaG, DwaaI, DwaaJ, DrfaH, DwbaP, DwaaL

Loss of oligosaccharide, increased sensitivity to DOC and polymyxin B, and human serum, highly attenuated, reduced invasiveness of host tissue, reduced spleen inflammation

Reduced immunogenicity compared to parent strain

[48]

lpxE

Fivefold reduction of virulence, low endotoxic activity in vivo and in vitro

Against pneumococcal challenge in mice

[50]

DpagP, msbB

Reduced virulence, sensitivity to polymyxin B, less inflammatory responses in vivo

Retained IgG levels against pneumococcal antigen

[51,52]

pagP

Increased reactogenicity in vitro

Increased protection against B. pertussis

[53,54]

pagL

Increased reactogenicity in vitro, increased LPS release from membrane, similar reactogenicity in vivo

Increased protection against B. pertussis

[53,54]

DlpxL1

Reduction of pro-inflammatory cytokines, 40-fold less pyrogenic in rabbits

Protective SBA titers against parent strain in mice, monkeys and humans

[59–63,66]

DlpxL2

Reduction of pro-inflammatory cytokines, 200-fold less pyrogenic in rabbits

One-third of the test subjects developed fourfold increase of SBA in humans

[67]

DlgtB, DlpxL1

lgtB deletion causes loss of the galactose sugar in the oligosaccharide, increased OMV internalization, enhanced DC maturation, increased Th17

Not tested

[71]

B. pertussis

pagL

Less toxicity than the wild type by reduced weight loss and reduced IL-6 induction in mice

Against intranasal challenge with B. pertussis

[65]

Vibrio cholerae

DmsbB

Reduced pro-inflammatory cytokines in RAW cell line, similar to wild type IgG1 and total IgG induction

Against V. cholerae O1

[68]

Shigella sonnei/ flexneri

DmsbB

800-fold reduction of inflammatory cytokines

Not tested

[69]

DhtrB

800-fold reduction of inflammatory cytokines, compensatory palmitoleoylation in S. flexneri

Not tested

[69]

DmsbB, DPagP

Less toxicity in vitro and in vivo, less induction of inflammatory cytokines, T cell adjuvant activity comparable to wild type in mice

Not tested

[74]

Salmonella spp.

[42,43]

Whole cell vaccines Bordetella pertussis

OMVs Neisseria meningitidis

Escherichia coli C10 C12 C14 C16 C18

GalN Phosphate group

DC: Dendritic cell; DOC: Deoxycholate; LPS: Lipopolysaccharide; OMV: Outer membrane vesicle; RAW cell line: A mouse macrophage cell line; SBA: Serum bactericidal antibody.

doi: 10.1586/14760584.2015.1026808




Vaccine applications of OMVs face the same questions as whole cell vaccines regarding the role of LPS, which on the one hand is beneficial as an intrinsic adjuvant, but on the other hand can be a potential problem due to endotoxic activity and reactogenicity. Detergent-treated OMVs using deoxycholate to remove most of the LPS have been used successfully to control meningococcal epidemics with efficacy rates of 70–83% in adults. These OMVs contain wild-type hexa-acylated LPS, but the detergent treatment reduces the amount of LPS, making them less toxic [14]. Some downsides of this method are reduction of membrane-bound lipoproteins that could be important vaccine components, such as factor H binding protein [73], and formation of aggregates, which influences the storage stability of OMV vaccines [14]. Another strategy for reducing OMVassociated endotoxicity is the use of genetically attenuated LPS. In the case of meningococcal OMV vaccines, the use of lpxL1 mutant strains carrying penta-acylated LPS has been investigated extensively [14,73–76]. The so-called native OMVs can be isolated from lpxL1 mutants without the need for any detergent extraction, and they display a level of endotoxic activity similar to detergent-extracted OMVs from strains with wild-type LPS while retaining lipoproteins such as factor H binding protein [77,78]. Furthermore, lpxL1 and lpxL2 native OMVs were safe and immunogenic in Phase I clinical studies [79,80]. Similar approaches have been taken for OMV vaccines against other pathogens, such as mutation of msbB in Vibrio cholerae [81] and deletion of msbB and htrB in Shigella species. The group working on Shigella OMVs refers to them as Generalized Modules for Membrane Antigens [82]. They have shown that the Generalized Modules for Membrane Antigens with penta-acylated lipid A cause considerable reduction in the induction of inflammatory cytokines, DhtrB by 800-fold and DmsbB by 300-fold. The residual activity of these Generalized Modules for Membrane Antigens is mainly by TLR2 activation and not mediated by LPS [82]. In the case of B. pertussis, OMVs from both wildtype and PagL-expressing strains have been investigated. Both showed protection against intranasal pertussis challenge in mice, but less toxicity was found for the PagL OMVs [83]. Although most LPS modifications introduced in OMVs are for the sole purpose of reducing toxicity of the vaccine, in meningococcal OMVs, an lgtB oligosaccharide mutation combined with lpxL1 showed enhanced OMV uptake and maturation by human dendritic cells (DCs) and increased Th17 cell expansion [84]. This enhanced uptake was attributed to the lgtB-truncated oligosaccharide’s ability to bind to human DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a C-type lectin involved in cell adhesion and antigen presentation to T cells [85]. There was no evidence of enhanced adjuvant effects for OMVs with lgtB LPS in a mouse immunization experiment [86]. However, differences in receptor specificity and expression pattern between murine and human DC-SIGN homologs complicate the interpretation of these results. OMVs from pathogens have been utilized as a vaccine, but they can also be applied as an adjuvant independent of their specific antigenic content. Penta-acylated OMVs prepared from informahealthcare.com

Review

non-pathogenic E. coli W3110 displayed similar adjuvant properties toward separately added keyhole limpet hemocyanin antigen, compared to both wild-type hexa-acylated OMVs and isolated LPS, as shown by antigen-specific IgG responses, DC activation markers and T cell proliferation. This penta-acylated OMV also induced far less toxicity in mice when compared to wild-type OMVs, including reduced induction of inflammatory cytokines (TABLE 3) [87]. OMVs are also included in the menB vaccine 4CMenB (Bexsero) developed by Novartis. 4CMenB consists of three purified recombinant proteins, and OMVs prepared with a detergent-based method. Clinical studies demonstrated that inclusion of OMVs in this vaccine resulted in greater immunogenicity and larger breadth of protection conferred by the individual recombinant proteins [88,89]. On this note, OMVs can also be used as a platform for direct expression of antigens, instead of just mixing them with separately purified recombinant proteins. This has the benefit of combining the intrinsic adjuvant properties and easy production method of OMVs compared to recombinant proteins [90–92]. Thus, LPS modifications can provide safe whole cell and OMV vaccines including LPS as an intrinsic adjuvant and potentially increase the uptake by DCs. Mutant LPS as isolated adjuvant

The main focus of research toward the use of LPS as an adjuvant has been to try and reduce toxicity while retaining its immunostimulatory activity. Several structural features of LPS influence its capabilities of inducing inflammatory responses through the TLR4/MD-2 complex. The dominant feature is the specific acylation and phosphorylation pattern of the lipid A moiety. Generally speaking, LPS containing six acyl chains is the most active, although there can be slight differences in activity between different hexa-acyl forms, for example, the lipid As from N. meningitidis and E. coli. This difference is due to the fact that N. meningitidis lipid A has symmetrical positioning of the fatty acid chains which are also shorter, making it a more potent inducer of TNF-a and IFN-b in mouse macrophages [93]. In the case of monophosphorylated penta-acylated LPS of Bacteroides fragilis and Porphyromonas gingivalis, there is up to 1000-fold difference in activating capacity, which is most probably also due to the length of certain acyl chains as well as the phosphorylation pattern [94]. Even a small change such as switching a single fatty acid of hexa-acylated E. coli LPS with a slightly longer one as found in P. gingivalis, or substitution of a fatty acid of N. meningitidis for a longer or shorter version, has direct impact on recognition by mono mac 6 cells, a human monocytic cell line with several features such as phagocytic ability and the ability to produce cytokines [95,96]. LPS preparations of a few bacterial species have been compared for the induction of MyD88-dependent and -independent (TRIF-dependent) cytokines. It seems that there is differential induction of these pathways, where N. meningitidis LPS is a potent inducer of both pathways in human macrophages, E. coli O55:B5 and V. cholerae LPS mainly induce the doi: 10.1586/14760584.2015.1026808


Review


MyD88-dependent pathway and Salmonella LPS preferentially activates the TRIF pathway [97]. In the 70s, the first attempts were made to chemically detoxify LPS from Salmonella minnesota, which normally contains up to seven acyl chains, three phosphates and a polysaccharide chain of varying length attached to a di-glucosamine head group. Sequential acid and alkaline hydrolyses of this LPS resulted in a mixture of acylated di-glucosamines with a major species of six acyl chains, no polysaccharide and only one phosphoryl group at the 4¢ position. This modified LPS is named MPLA. MPLA has reduced toxicity as measured in vitro through the release of pro-inflammatory cytokines by innate immune cells, while retaining a clear adjuvant effect. In animal models, MPLA showed no adverse effects and also based on clinical studies, it was considered a safe adjuvant [16,98]. MPLA is the first approved TLR agonist to be used as an adjuvant and the first new adjuvant for human use in over 70 years. One of the main reasons that MPLA has reduced toxicity while retaining immunostimulatory activity is thought to be its reduced signaling through MyD88 while retaining signaling through the TRIF pathway [99]. The TRIF pathway plays a more prominent role than MyD88 in allowing TLR4 agonistmediated stimulation of early T cell responses, and in an adoptive T cell transfer model, TRIF deficiency prevented a lipid A agonist from enhancing T cell proliferation and survival [100]. In addition, during priming of DCs, upregulation of costimulatory molecules CD86 and CD40 is dependent on TRIF whereas CD80 is dependent on both MyD88 and TRIF [100]. Some bacterial species naturally express a less toxic LPS, making these strains possible candidates for whole cell vaccines with intrinsic adjuvant activity or as source for isolated LPS for use as adjuvant. For example, Bacteroides thetaiotaomicron and Prevotella intermedia naturally express a lipid A structure that is both monophosphorylated and penta-acylated, similar to MPLA. Although isolated LPSs from these strains induce decreased MyD88 and TRIF-dependent cytokine responses compared to more standard LPS forms like from E. coli, they still stimulate antigen-specific antibody responses [101]. Another way of detoxifying LPS is by genetic modification. This can be achieved by inactivation or modification of genes involved in lipid A biosynthesis, in particular, genes involved in the addition of secondary acyl chains, because in contrast to earlier steps of the biosynthesis pathway, they are generally not essential for bacterial viability. LpxL1 and LpxL2 each add a secondary lauroyl residue in N. meningitidis, and their inactivation results in viable strains producing a penta-acylated lipid A structure [26]. Immunization of mice with OM complexes of an LPS-deficient N. meningitidis strain in combination with LpxL1 or LpxL2 mutant LPS showed in the case of LpxL1, equal adjuvant activity as wild-type LPS, while reducing the induction of TNF-a in a human monocytic cell line [26]. However, later studies showed that LpxL1 mutant LPS has a much stronger reduction of human versus murine TLR4/ MD-2 activation, making its relevance as adjuvant in humans uncertain [102]. doi: 10.1586/14760584.2015.1026808

Additional options for lipid A engineering are provided by the many LPS modification enzymes which have been described from several bacterial species (TABLE 2). For instance, PagL has been shown to remove the 3-O-linked acyl chain of lipid A in B. pertussis [68], whereas LpxE removes the phosphate group from the 1¢-position of lipid A in F. novicida [103]. The modifying enzymes that a bacterial species naturally contains are diverse. The gene encoding the enzyme PagL is present in B. bronchiseptica, but is lacking in N. meningitidis. However, PagL can be introduced in N. meningitidis through cloning and heterologous expression from an inducible promoter resulting in efficient removal of the 3-O-linked acyl chain. Pentaacylated LPS isolated from this strain induces much lower amounts of MyD88-dependent cytokine IL-6 compared to wild-type hexa-acylated LPS, but retains most of its capacity to induce the TRIF-dependent chemokine IP-10 [33]. As both LpxL1- and PagL-altered LPSs from the respective meningococcal mutants are penta-acylated but display different agonist properties, these results highlight the importance of the specific acylation pattern versus just the total number of acyl chains in determining activity. Vaccination against B. pertussis has changed in many countries from using a whole cell pertussis vaccine to a more defined acellular pertussis vaccine. Because acellular pertussis vaccines do not contain LPS, they are less reactogenic, but they are also less immunogenic and induce a poorer memory response. The inclusion of Salmonella-derived MPLA or LpxL2 mutant LPS from N. meningitidis to an acellular pertussis vaccine resulted in higher immune responses and better protection against pertussis in mice, while the induction of serum IL-6 levels was substantially lower than with whole cell pertussis [10]. A combination of inactivation of lipid A biosynthesis genes and expression of genes encoding LPS-modifying enzymes gives the opportunity to remodel lipid A into the desired structure [13]. This, in turn, allows controlling the amount of inflammatory cytokines produced by innate immune cells while retaining the adjuvant activity. With genetic modification, it is also possible to create strains directly making MPLA without the need for further chemical treatment. This has been done in S. typhimurium and E. coli by a combination of genetic modifications including induction of the enzyme LpxE [12,13,104,105]. Role of (core-)oligosaccharide

The core-oligosaccharide of LPS typically consists of six to eight sugar moieties covalently linked to Kdo-lipid A. Among bacterial species, the core-oligosaccharide is more variable than the lipid A, and especially in the outer core, substantial differences can be found even among strains within a single species. Besides sugar residues, the core can include non-carbohydrate components such as phosphates, phosphoethanolamines and amino acids. Although the lipid A part of LPS is the main determinant for the interaction with the TLR4/MD-2 receptor complex, a study on Salmonella LPS showed that isolated lipid A has far Expert Rev. Vaccines



lower TLR4 activating capacity than the complete LPS molecule. This difference was attributed to the contribution of Kdo residues, because Re LPS consisting of lipid A-Kdo showed increased activity compared to lipid A alone [106]. The isolated hexa-acylated lipid A from N. meningitidis also showed strongly reduced activity as compared to complete LPS [107]. On the same note, lipid A from Capnocytophaga canimorsus, a normal member of mouth flora in dogs, also induced far less endotoxic activity compared to lipid A including core-oligosaccharide. Apparently, in this case, the core-oligosaccharide plays an unexpected role in the binding to MD-2, which is not mediated by Kdo but a more distal region [108]. A study by Bergman et al. revealed a role of the oligosaccharide in immune suppression. Binding of the O antigen of Helicobacter pylori LPS to DC-SIGN resulted in blocking of Th1 development and decreased IL-6 production and increased IL-10 production in DCs [109]. In contrast, an lgtB LPS mutant of N. meningitidis is able to bind to DC-SIGN, and thereby enhances phagocytosis and induces Th1 skewing [85]. Furthermore, LPS oligosaccharide carrying a terminal N-acetylgalactosamine of N. gonorrhoeae interacts with the macrophage galactose lectin and induces skewing toward a Th2-type response [110]. Thus, binding of LPS oligosaccharide to C-type lectins such as DC-SIGN can modulate the immune response in either direction. The lgtB truncation of LPS in N. meningitidis results in expression of a core-oligosaccharide with a terminal GlcNAc residue, which must be important for the interaction with DC-SIGN as the parent strain does not bind [85]. In addition, a study by Zhang et al. using an array of mutant LPS strains of several bacteria capable of binding DC-SIGN showed that this was consistently related to the presence of GlcNAc [111]. However, glycan arrays used to determine DC-SIGN oligosaccharide specificity did not present GlcNAc as a DC-SIGN ligand, and therefore, interaction of GlcNAc with DC-SIGN also depends on the presence of the backbone oligosaccharide [112]. Besides DC-SIGN, other host receptors for coreoligosaccharide structures in LPS undoubtedly exist. Brain angiogenesis inhibitor 1 is a transmembrane receptor involved in inhibition of angiogenesis and apoptosis by binding to phosphatidylserine [113]. In a recent study, the brain angiogenesis inhibitor 1 receptor was shown to bind to the coreoligosaccharide of LPS from E. coli, S. typhimurium and Campylobacter jejuni, thereby mediating binding and engulfment of gram-negative bacteria by macrophages [114]. Furthermore, binding of this newfound pattern recognition receptor by LPS enhanced the TNF-a production in macrophages [114]. It is not yet clear which part of the core-oligosaccharide is responsible for binding to the brain angiogenesis inhibitor 1 receptor. Potentially the combination of an LPS oligosaccharide mutant, such as lgtB that targets DCs, with an antigen could enhance uptake of the antigen and induce a more effective immune response. Inclusion of the core-oligosaccharide in LPS to be used as adjuvant also has the additional benefit of making the adjuvant water soluble as compared to just using the highly informahealthcare.com

Review

hydrophobic lipid A, which can simplify formulation issues. Of course, inclusion of core-oligosaccharide also introduces the possibility of inducing an antibody response against it, although this was found to be very limited in the case of purified meningococcal LPS mutants [115]. The fact that certain LPS species with mutant oligosaccharide possess the ability to skew the immune response toward either Th1 or Th2, enhance endotoxic activity through increased induction of proinflammatory cytokines, or potentially increase uptake by DCs by targeting receptors such as DC-SIGN makes it relevant to include the core-oligosaccharide structure when investigating LPS-based adjuvant activity. Expert commentary

It has been suggested that the most suitable LPS-derived adjuvant still activates the TRIF pathway equal to wild-type LPS, but has reduced MyD88 pathway activation. This view is supported by the fact that the approved adjuvant MPLA was shown to mainly activate the TRIF pathway [99]. Strong activation of the MyD88 pathway can result in unwanted, potentially harmful inflammatory responses. It is well established that membrane-bound LPS causes less inflammatory responses and has lower endotoxic activity, compared to soluble LPS [116,117]. This may be caused by reduced accessibility of LPS embedded in the OM to LPS receptors such as LPS binding protein, CD14 and TLR4/MD-2, or the need for membrane-bound LPS to be processed by macrophages resulting in a mainly intracellular presentation. One way of trying to target the TRIF pathway is through modification of the LPS, as discussed above. However, another way is the use of different formulations of LPS, such as liposomes or other types of particles. Liposomes give the ability to encapsulate proteins and/or LPS inside lipid vesicles, and they can deliver LPS into endosomes through endocytosis, thereby only initiating the TRIF pathway. Recent studies have shown that LPS encapsulated in liposomes does not induce inflammatory cytokines including IL-6 and TNF-a from DCs, but enhances Th1 responses by increased induction of type I interferons, which is consistent with the idea that only the TRIF pathway is targeted in this way [118]. A crucial problem with TLR4 stimulatory adjuvants like LPS is the lack of good animal models. TLR4/MD-2 activation by LPS derivatives can be highly species specific. For example, lipid IVa, the minimal LPS precursor structure of E. coli, behaves as an antagonist with human TLR4/MD-2, but acts as an agonist in mice. Studies of the crystal structure of TLR4/ MD-2 interacting with lipid IVa have revealed differences in the lipid IVa–TLR4/MD-2 complex formation between the human and murine receptors [34]. A transgenic mouse strain expressing the human TLR4 and MD-2 receptors appears to be a promising new animal model for in vivo testing of LPS activity [119]. In addition, other species might also provide more suitable animal models than mice, although there have not yet been many comparative studies investigating them for testing of LPS adjuvants. The common use of rabbits in LPS doi: 10.1586/14760584.2015.1026808


Review


pyrogenicity studies underscores the importance of knowing differential species recognition of LPS variants. Another consideration when studying LPS as an adjuvant is the substantial heterogeneity of LPS preparations purified from bacterial strains and the influence of the isolation and purification methods used. Especially when using LPS modifying enzymes for removal of certain molecular forms, such as LpxE to remove a phosphate group or PagL for 3-O deacylation, there is no guarantee that these enzymes are 100% efficient; so, the end result may be a mixture of different LPS species. Such mixtures may make it difficult to see the full effect on the modified LPS, as small amounts of the highly active wild-type LPS can still have a major influence. Therefore, some studies have been directed toward purifying a homogeneous LPS structure, and testing the difference after purification steps combined with careful analysis of all molecular species present [33]. Recently, intracellular recognition of LPS was observed independent of the TLR4/MD-2 receptor and the entire TLR4–TRIF-type I interferon pathway [120]. Cytoplasmic LPS can trigger the non-canonical inflammasome requiring caspase11. Caspase-11 knockout mice survive a lethal dose of LPS similar to TLR4 knockout mice, making caspase-11 a key player in the in vivo toxicity of LPS and septic shock. Recently, Shi et al. have discovered that caspase-11 or the homolog in humans, caspase-4, is an innate immune receptor for cytoplasmically delivered LPS [121]. Similar to LPS recognition by TLR4, caspase-11 could also bind the TLR4 antagonist lipid IVa, which then fails to induce its oligomerization and activation [121]. What effect this mode of LPS recognition

has with other LPS modifications and how it impacts upon their use as adjuvants is not clear and needs further investigation. Five-year view

In the coming years, genetic LPS modifications will certainly give rise to several potential new adjuvants. It has already been demonstrated that MPLA can be produced more easily by the use of genetic modification of bacterial strains instead of chemical treatment. New isolation and purification techniques will also allow the development of more pure LPS extractions, thereby reducing any side effects caused by contaminations. A major hurdle is the safety issues, but a detailed understanding of the underlying molecular mechanisms for LPS-based adjuvant activity as well as reactogenicity will make it easier to predict which animal models are indicative for the expected performance in humans. The use of LPS-containing vaccines such as those based on OMVs is currently receiving a lot of attention, and this approach will benefit greatly from the use of LPS mutants with lower endotoxic activity. Such vaccines have already entered the stage of clinical trials for meningococci, and others are in advanced preclinical development. Financial & competing interests disclosure

The authors were supported by Intravacc. P van der Ley is an employee of Intravacc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues .

Gram-negative bacteria possess lipopolysaccharide (LPS) as a major component of their outer membrane. The lipid A portion of LPS binds to the pattern recognition receptor toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), leading to innate immune signaling. Many pathogenic bacteria can modify their LPS structure to evade this immune recognition and/or resist antimicrobial peptides.

.

Biosynthetic engineering of LPS through deletion of LPS biosynthesis genes or induction of modifying enzymes can reduce its endotoxicity while retaining adjuvant activity, resulting in safer live attenuated vaccines, whole cell vaccines and outer membrane vesicle (OMV) vaccines.

.

lpxL1 deletion in Neisseria meningitidis has provided a safe OMV vaccine, as shown by a Phase I clinical trial, with reduced endotoxicity while retaining potentially immunogenic outer membrane components. N. meningitidis lgtB mutant oligosaccharide interacts with dendritic cell-specific ICAM-3-grabbing non-integrin, leading to increased uptake and maturation of OMVs by human dendritic cells.

.

The combinatorial use of LPS-modifying enzymes in Escherichia coli and other bacteria provides an immense amount of potential LPS structures with different immune-activating properties that can be explored as adjuvants.

.

Animal models for testing the adjuvant activity of modified LPS are complicated by the species-specific recognition of different LPS forms by TLR4/MD-2. The construction of a human TLR4/MD-2 transgenic mouse has created a better animal model for testing LPSbased adjuvants. Alternatively, in vitro models using TLR4/MD-2 expressed from different species can be used to predict suitable in vivo models for a specific application.

.

Caspase-11 is a newfound pattern recognition receptor for binding cytoplasmic LPS. Deletion of this receptor in mice makes them less susceptible for sepsis after administration of LPS. Its potential role in LPS adjuvant activity needs to be explored.

.

Future research will give more insight in different LPS structures and their immune activating potential. New purification methods for preparing homogeneous LPS structures will help determine the full potential of certain modifications made to LPS.

doi: 10.1586/14760584.2015.1026808



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