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Modified hexa-acylated neisserial LPS

20220281907 · 2022-09-08

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Abstract

The present invention relates to neisserial LPS having a hexa-acylated lipid A moiety, wherein the hexa-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it comprises a palmitoleoyl instead of a lauroyl secondary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. The invention further relates to mixtures of the hexa-acylated LPS with the corresponding penta-acylated LPS, lacking a secondary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. The invention also relates to neisserial bacteria that have been genetically modified to reduce expression of the endogenous 1pxL1 gene and to introduce expression of a heterologous thermosensitive 1pxP gene for producing the hexa- and penta-acylated LPS. By selecting the time and/or temperature at which the bacterium is grown, it is feasible to increase or decrease the amount of hexa-acylated lipid A structure relative to the corresponding penta-acylated structure and thereby modulate the TLR4 agonist activity of the neisserial LPS of the invention, to the exact level of activity required for a particular immunotherapeutic approach.

Claims

1. A genetically modified bacterium of the genus Neisseria, wherein the bacterium comprises: a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and, b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity.

2. The genetically modified bacterium according to claim 1, wherein the bacterium is a genetically modified Neisseria meningitidis, Neisseria gonorrhoeae or Neisseria lactamica.

3. The genetically modified bacterium according to claim 1, wherein the genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity is a genetic modification that introduces the expression of a heterologous lpxP gene.

4. The genetically modified bacterium according to claim 3, wherein the heterologous lpxP gene has a nucleotide sequence that encodes an LpxP lipid A palmitoleoyltransferase that has at least 80% amino acid sequence identity with at least one of SEQ ID NO's: 4-10.

5. The genetically modified bacterium according to claim 3, wherein the nucleotide sequence encodes a LpxP palmitoleoyl acyltransferase having at least 85% amino acid sequence identity with at least one of SEQ ID NO: 4-10.

6. The genetically modified bacterium according to claim 3, wherein the nucleotide sequence encodes a LpxP palmitoleoyl acyltransferase having at least 90% amino acid sequence identity with at least one of SEQ ID NO: 4-10.

7. The genetically modified bacterium according to claim 1, wherein the endogenous lpxL1 gene is a gene encoding an LpxL1 protein having an amino acid sequence with at least 90% sequence identity with at least one of SEQ ID NO's: 1-3.

8. The genetically modified bacterium according to claim 1, wherein the bacterium is further genetically modified to express a heterologous antigen.

9. The genetically modified bacterium according to claim 8, wherein the heterologous antigen is expressed on the extracellular outer membrane surface of the bacterium.

10. The genetically modified bacterium according to claim 1, wherein the bacterium has a genetic modification that reduces or eliminates the expression of at least one of an endogenous lgtB gene and an endogenous galE gene.

11. The genetically modified bacterium according to claim 1, wherein the bacterium is a Neisseria meningitidis serogroup B, immunotype L3.

12. The genetically modified bacterium according to claim 1, wherein the bacterium is Neisseria meningitidis strain H44/76 or a derivative thereof.

13. A neisserial LPS having a hexa-acylated lipid A moiety, wherein the hexa-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it has a palmitoleoyl (instead of a lauroyl) as secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety.

14. The neisserial LPS according to claim 13, wherein the LPS is obtainable from a genetically modified bacterium of the genus Neisseria, wherein the bacterium comprises: a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and, b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity.

15. The neisserial LPS according to claim 13, wherein, except for the hexa-acylated lipid A moiety, the LPS has the structure of LPS of Neisseria meningitidis, Neisseria gonorrhoeae or Neisseria lactamica.

16. The neisserial LPS according to claim 13, wherein the Neisseria meningitidis, Neisseria gonorrhoeae or Neisseria lactamica is at least one of lgtB- and galE.

17. The neisserial LPS according to claim 13, wherein the Neisseria meningitidis is at least one of serogroup B and immunotype L3.

18. The neisserial LPS according to claim 13, wherein the hexa-acylated lipid A moiety has the structure of formula (I): ##STR00003## wherein R.sub.1 and R.sub.2, independently, are either —P(O)(OH).sub.2, —[P(O)(OH)—O].sub.2—H, —[P(O)(OH)—O].sub.2—CH.sub.2CH.sub.2NH.sub.2, —[P(O)(OH)—O].sub.3—CH.sub.2CH.sub.2NH.sub.2, —[P(O)(OH)—O].sub.3—H or —P(O)(OH)—O—CH.sub.2CH.sub.2NH.sub.2.

19. An OMV comprising the neisserial LPS according to claim 13.

20. The OMV according to claim 19, wherein the OMV is obtainable from a genetically modified bacterium of the genus Neisseria, wherein the bacterium comprises: a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and, b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity.

Description

DESCRIPTION OF THE FIGURES

[0095] FIG. 1A. FIG. 1B. FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G. FIG. 1H FIG. 1I, FIG. 1J, FIG. 1K, FIG. 1L. Charge deconvoluted ESI-FT mass spectra of LPS. The charge deconvoluted ESI-FT mass spectra of the LPS isolated from twelve different strains of Neisseria meningitidis are shown as follows: parent HB-1 strain FIG. 1A, ΔlpxL1 FIG. 1B, ΔlpxL2 FIG. 1C, pagL FIG. 1D, ΔlpxLJ-pagL FIG. 1E, ΔlpxL2-pagL FIG. 1F, ΔlpxL1-lpxP cultured at 30° C. for 5 h FIG. 1G or at 25° C. overnight FIG. 1H, ΔlptA FIG. 1I, ΔlptA-ΔlpxL1 FIG. 1J, ΔlptA-pagL FIG. 1K and ΔlptA-lpxE FIG. 1L. A simplified representation of the LPS structure assigned to the ion of 3408.507 u is included in mass spectrum FIG. 1A. The vertical line at a mass of 3408.514 u, which corresponds to the calculated molecular mass of this latter LPS species, is used as a reference to indicate LPS composition assigned to other ion signals. See Supplemental Table 1 for detailed LPS composition proposals. All annotations refer to monoisotopic masses of the neutral molecules.

[0096] FIG. 2A-FIG. 2B TLR4 activation by N. meningitidis strains as indicated. HEK-blue hTLR4 cells were stimulated with 5-fold serial dilutions of heat-inactivated N. meningitidis for 20 h. TLR4 activation was measured by detection of secreted alkaline phosphatase. Results of serial dilutions are depicted in a line graph FIG. 2A and for a single OD.sub.600nm of 0.0004 in a bar graph FIG. 2B. Data are expressed as mean values or mean+SD of three independent experiments. Statistical significance was determined with an ANOVA test comparing against HB-1. *, p<0.05; ****, p<0.0001. Data are also presented in Table 5.

[0097] FIG. 3. TLR4 activation by purified LPS structures. HEK-blue hTLR4 cells were stimulated with 10-fold serial dilutions of 12 different LPS structures. TLR4 activation was measured by detection of secreted alkaline phosphatase. Data are representative results from three independent experiments and are depicted as the mean values of triplicates.

[0098] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G. Cytokine release of MM6 cells stimulated with purified LPS structures. MM6 cells were incubated with 10-fold serial dilution of different LPS structures for 20 h. IL-6 FIG. 4A, IP-10 FIG. 4B, IL-1p FIG. 4C, MCP-1 FIG. 4D production was measured by ELISA. IL-6 and IL-1p are considered MyD88 dependent cytokines and IP-10 and MCP-1 are more TRIF dependent. Cytokine levels of MM6 cells stimulated with 5 ng/ml LPS are also presented as percentages of the HB-1 strain FIG. 4E and cytokine ratios in concentration FIG. 4F and percentages FIG. 4G. For the cytokine ratios (FIG. 4F+FIG. 4G) the background without LPS stimulation was subtracted. Data shown are depicted as the mean values of two independent experiments. Statistical significance was determined with a 2-way ANOVA test comparing against HB-1. *, p<0.05. Data are also presented in Tables 6 and 7.

EXAMPLES

1. Methods and Materials

[0099] 1.1 Bacterial Strains and Plasmids All mutants were created in a N. meningitidis H44/76 strain (HB-1) strain using plasmid pMF121, resulting in deletion of the capsular biosynthesis locus including the galE gene. N. meningitidis strains were grown on GC medium base (Difco) plates supplemented with IsoVitaleX, in a humid atmosphere containing 5% C02 at 37° C. For liquid culture, strains were grown in 36 mg/mL tryptic soy broth medium (Difco) in a conical flask at 37° C., shaken at 140 RPM. Required antibiotics were added to plate and liquid cultures (kanamycine 100μg/ml, chlooramphenicol 3μg/ml). The lpxL1 and lpxL2 mutants were obtained by transformation with a linearized PCRII plasmid (Invitrogen) carrying the genes with a kanamycine resistance cassette described by van der Ley et al. (15) or a pGem T easy plasmid (Promega) with the lpxL1 gene that has a deleted section replaced with a chloramphenicol (CAM) cassette. For the lptA mutant the gene was amplified by PCR from the H44/76 strain, cloned into a pGem T easy plasmid (Promega) and a kanamycine cassette was placed in the gene at the MunI restriction site. The plasmid was linearized by digestion with a restriction enzyme cleaving outside the gene and transformed into the N. meningitidis H44/76 (HIB-1) strain. N. meningitidis derivatives carrying the genes pagL, lpxP and lpxE were created using a pEN11 plasmid previously described for the expression of the Bordetella bronchiseptica pagL gene (13, 18). To obtain lpxP and lpxE derivatives the pagL gene in the pENI1 plasmid was replaced with the lpxP or lpxE gene amplified by PCR from E. coli and B. bronchiseptica, respectively. Expression of the genes on the pen1 1 plasmid was induced by addition of 1 mM isopropyl-β-D-thioglactopyranoside (IPTG) and CAM (3μg/ml) to the liquid culture medium. Primers are listed in Table 1.

TABLE-US-00001 TABLE 1 PCR primers used in the construction of the mutant strains SEQ Primer Sequence (5′-3′) Source ID NO LptA Fw GCCTTCCTTTCCCTGTATTC N. meningitidis LptA Re GGTGTTCGGACACATATGC N. meningitidis LpxL1 Fw CTGATCGGGCAGATACAG N. meningitidis LpxL1 Re GTGCGCTACCGCAATAAG N. meningitidis LpxL2 Fw AAACAGATACTGCGTCGGAA N. meningitidis LpxL2 Re CCCTTTGCGAACCGCCAT N. meningitidis PagL Fw ATGCAATTTCTCAAG B. bronchiseptica PagL Re TCAGAACTGGTACGT B. bronchiseptica LpxP Fw CATATGGCCGCTTACGCAGACAATACAC E. coli LpxP Re GACGTCACGCCTGAATGACTTCATTACACC E. coli LpxE Fw CATATGATCCGGCCCTCATCCCATTCCC B. bronchiseptica LpxE Re TCATGACCCGAAAGGCGCTTCCCTTCAG B. bronchiseptica

1.2 LPS Isolation

[0100] LPS from bacterial mutants was extracted with hot phenol-water (19) and purified further by solid phase extraction (SPE) on reverse phase cartridges. In short, cells from 50 ml of bacterial culture with an OD.sub.600nm of 1.4 (or 100 ml of the ΔlpxL1-lpxP mutant grown at 30° C.) were collected by centrifugation at 2,739×g for 1 h at 20° C. Then, bacteria were suspended in 20 ml of water and centrifuged at 2,739×g for 25 min at 20° C. For hot phenol-water extraction, bacterial pellets were suspended with 4 ml of water, heated to 70° C., mixed with 3.2 ml of phenol at the same temperature and kept under agitation for 10 min at 70° C. The aqueous phase was separated from the phenolic phase by centrifugation at 2,739×g for 15 min at 20° C. After transferring the aqueous phase to a new vial, the phenolic phase was extracted again by adding 3 ml of water at 70° C. and repeating the extraction procedure. The aqueous phases from two consecutive extractions were pooled (˜6.5 ml) and prepared for SPE by adding 5 ml of 0.356 M triethylammonium acetate (TEAA) pH 7 (solvent A) and 3.8 ml of 2-propanol:water:triethylamine:acetic acid (70:30:0.03:0.01, v/v) pH 8.7 (solvent B). In total, ten LPS extracts each from a different bacterial mutant could be purified simultaneously by SPE on reverse phase Sep-Pak C18 cartridges (1 ml syringe-barrel-type Vac cartridge, 50 mg of C18 resin, Waters) using a 20-position vacuum manifold (Waters). Cartridges were conditioned for SPE by applying consecutively 1 ml of 2-propanol:water:triethylamine:acetic acid (85:15:0.015:0.005, v/v) pH 8.7 (solvent C), 0.07 mM TEAA pH 7 (solvent D) and solvent A under vacuum. Then, samples were split into two aliquots of equal volume and each aliquot was applied into a different cartridge. Next, cartridges were washed once with 1 ml of solvent A and twice with 1 ml of 20% (v/v) solvent B in solvent D. LPS was eluted from the columns by applying 0.6 ml of solvent C. Eluates from the same sample were combined (1.2 ml per sample in total) and dried in a centrifugal vacuum concentrator (Concentrator plus, Eppendorf) at room temperature. LPS concentration in isolated samples was determined by the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) assay (20). In addition, the purity and integrity of purified samples were judged by Tricine-SDS-PAGE using 1 mm-thick, 16% precast Novex© mini-gels (Thermo Fisher Scientific Inc.), LPS silver staining (21) and protein visualization with Imperial™ Protein Stain (Thermo Scientific). 1.3 Mass spectrometry Electrospray ionization Fourier transform mass spectrometry (ESI-FT-MS) was performed on an LTQ Orbitrap XL instrument (Thermo Scientific) in negative ion mode.

[0101] LPS samples were dissolved in a mixture of water, 2-propanol and triethylamine (50:50:0.001, by volume) pH 8.5 and infused into the mass spectrometer by static nano-ESI (22, 23). The MS instrument was calibrated with a Pierce Negative Ion Calibration Solution (Thermo Scientific) and internally with taurocholic acid following standard procedures provided by the manufacturer (Thermo Scientific). Fragmentation analysis of intact LPS was carried out by in-source collision-induced fragmentation (SID). Y- and B-type fragment ions, corresponding to the lipid A and oligosaccharide moieties of LPS, respectively, were generated by SID at a potential difference of 100 V. Fragment ions are annotated according to the nomenclature of Domon and Costello (24). Mass spectra were charge-deconvoluted using the Xtract tool of Thermo Xcalibur 3.0 software (Thermo Scientific). All mass values given refer to monoisotopic molecular masses.

[0102] Proposed LPS compositions are based on the general chemical structure of the L3 immunotype LPS from N. meningitidis reported previously (25, 26).

1.4 Cell Stimulation Mono Mac 6 cells were seeded at 1×10.sup.5 cells per well in 96 well microtiter plates in 100μl Iscove's modified Dulbecco's medium (IMDM) (Invitrogen) medium supplemented with 100 units/ml penicillin, 100μg/ml streptomycin, 292μg/ml 1-glutamine (Invitrogen), and 10% fetal calf serum (Invitrogen). Hek blue-hTLR4 cells (Invivogen), a HEK293 cell line stably expressing human TLR4, MD-2 and CD14, were seeded at 3.5×104 cells per well in 96-well microtiter plates in 100μl DMEM (Invitrogen) medium supplemented with 100 units/ml penicillin, 100μg/ml streptomycin, 292 μg/ml 1-glutamine (Invitrogen), and 10% fetal calf serum (Invitrogen). Cells were stimulated with 10-fold serial dilutions of LPS in IMDM (MM6 cells) or DMEM (HEK blue-hTLR4 cells) for 18-20h at 37° C. in a humid atmosphere containing 5% CO2. HEK-blue-hTLR4 cells were also stimulated with serial dilution of whole bacterial cells. Cytokine concentration in the supernatants of MM6 cells was determined by enzyme-linked immunosorbent assay (ELISA). All cytokine (IL-6, IL-1β, IP-10, MCP-1) concentrations were determined using a DUOset ELISA development kit (R&D systems) following the manufacturer's instructions. To quantify alkaline phosphatase secreted by HEK-blue-hTLR4 cells, 20μl of the supernatant from each well was added to 200μl Quanti-blue (Invivogen) and incubated at 37° C. for 2-3 hours. Read out was done on a spectrophotometer at 649 nm. Statistically significant differences were determined by the one-way (alkaline phosphatase secretion) or two-way (Cyokine release) ANOVA test by using GraphPad Prism 6.04 statistical software (GraphPad Software, Inc.).

2. Results

2.1 Bioengineering of Modified LPS Structures

[0103] LPS mutants in N. meningitidis were constructed in the HB-1 derivative of strain H44/76. The HB-1 strain is capsule deficient and has a galE deletion that results in truncation of the LPS oligosaccharide. Mass spectrometric analysis demonstrated that the HB-1 strain expresses a hexa-acylated, tri-phosphate, bis-phosphoethanolamine lipid A structure (see below). To construct a diverse set of LPS mutants in strain HB-1, we inactivated the autologous genes encoding for the LPS enzymes LptA, LpxL1 and LpxL2 and heterologously expressed the LpxE, LpxP and PagL LPS enzymes (see Table 2) by cloning the genes on the pen11 plasmid behind a lac promotor.

TABLE-US-00002 TABLE 2 Overview of the inactivated the autologous genes encoding for the LPS enzymes LptA, LpxLl and LpxL2 and heterologously expressed the LpxE, LpxP and PagL LPS enzymes. Source Enzyme Abbr. Activity organism LpxL1 L1 Adds C.sub.12 to the primary Neisseria linked acyl meningitidis chain at 2′-position LpxL2 L2 Adds C.sub.12 to the primary Neisseria linked acyl meningitidis chain at 2-position LpxE E Removes 1 phosphate Bordetella group bronchiseptica LpxP Lp Adds palmitoleate to the Escherichia coli primary linked acyl-chain at 2′-position. PagL P Removes acyl-chain from Bordetella 3-position bronchiseptica LptA La Adds phosphoethonalamine Neisseria groups at meningitidis 1 or/and 4′-position

[0104] In addition, combinations of deletion of autologous genes and expression of heterologous enzymes were constructed. This approach resulted in 11 LPS mutant strains as listed in Table 3.

[0105] For the expression of LpxE (Protein ID: CAE41138.1) we initially cloned an lpxE homologue from Bordetella pertussis. However, expression of the gene in HB-1 or its lptA mutant derivative did not result in any LPS structural changes as determined by mass spectrometry. As an alternative the lpxE (Genbank accession number: WP_003809405.1) homologue from Bordetella bronchiseptica, which exists as a pseudogene in B. pertussis, was cloned and expressed in a ΔlptA mutant strain. This resulted in the loss of a phosphate group in the lipid A and was included in our panel of LPS mutant strains (FIG. 1L).

[0106] LpxP (Genbank accession number: U49787.1), an enzyme known to add a secondary 9-hexadecenoic acid (C16:1) to the 2′ acyl chain in E. coli (27), was expressed in the N. meningitidis ΔlpxL1 mutant strain, because the LpxL1 enzyme also adds a secondary acyl chain on the same position. This modification was done to create a hexa-acylated lipid A structure different from the original by carrying a longer C16 secondary acyl chain in the 2′ position instead of C12. When LpxP was expressed in the ΔlpxL1 mutant strain at 37HC this resulted in a very faint addition of C6:1. However, the C16:1 is added onto E. coli LPS only at 12° C., so for this reason we grew the bacteria at lower temperatures. Cultivation of meningococci below 25C is, unlike in E. coli, not possible, but at 25° C. and 30° C. we already found a much higher relative abundance of the LpxP hexa-acylated lipid A structure carrying the additional C16:1, with 25° C. resulting in the highest efficiency (at least 50% relative abundance) (FIG. 11H).

TABLE-US-00003 TABLE 3 Overview of the constructed LPS mutants in the N. meningitidis HB-1 strain Acyla- Phosphoryla- Phospho- Strain Abbr. tion tion ethanolamine HB-1 parent strain Hexa Tris Bis ΔlpxL1 ΔL1 Penta Tris Bis ΔlpxL2 ΔL2 Penta Bis Mono pagL P Penta Tris Bis ΔlpxL1-pagL ΔL1-P Tetra Tris Bis ΔlpxL2-pagL ΔL2-P Tetra Bis Mono ΔlpxL1-lpxP 37° C. ΔL1-Lp37 Hexa Tris Bis ΔlpxL1-lpxP 30° C. ΔL1-Lp30 Hexa Tris Bis ΔlpxL1-lpxP 25° C. ΔL1-Lp25 Hexa Tris Bis ΔlptA ΔLa Hexa Tris None ΔlptA-ΔlpxL1 ΔLa-ΔL1 Penta Tris None ΔlptA-pagL ΔLa-P Penta Tris None ΔlptA-lpxE ΔLa-E Hexa Tris None

2.2 Mass Spectrometric Characterization of Modified LPS

[0107] The charge-deconvoluted ESI-FT mass spectra of intact LPS isolated from the constructed N. meningitidis mutants are shown in FIG. 1. The mass spectrum of LPS of the HB-1 (galE) parent strain (FIG. 1A), displayed an ion signal of 3408.507 u consistent with LPS comprised of wild-type hexa-acyl lipid A carrying three phosphate (P) and two phosphoethanolamine (PEA) groups and an L3-immunotype oligosaccharide structure substituted with a glycine (Gly) residue and truncated at the proximal galactose (Gal) of its alpha chain due to inactivation of the galE gene (Mcalc.=3408.514μ, see Supplemental Table 1 for LPS composition proposals). Accompanying ion peaks of 3351.488, 3285.501 and 3228.480 u (FIG. 1A) corresponded to LPS species which lack Gly (Ameas.=−57.019μ), carry one less PEA group in the lipid A (Ameas.=−123.006μ) or both (Ameas.=−180.027μ), respectively. This chemical heterogeneity of the LPS from HB-1 (galE-) strain is likely caused by variation in lipid A phosphorylation and oligosaccharide non-stoichiometric substitution with glycine. Composition proposals based on mass spectra of intact LPS were additionally supported by FT-MS analysis of LPS fragment ions corresponding to lipid A and oligosaccharide moieties, which were generated by in-source collision induced dissociation (SID) of intact LPS. For instance, SID FT mass spectra of LPS from HB-1 (galE-) strain displayed fragment ions of 1916.098 and 2039.106 u corresponding to hexa-acyl lipid A species with 2 and 3 PEA groups (Mcalc.=1916.100 and 2039.109μ, respectively) and a fragment ion of 1369.404 u corresponding to the dehydrated derivative of the oligosaccharide moiety described above (Mcalc.=1369.406μ). Fragmentation analyses of LPS derived from other strains of N. meningitidis described here showed that different types of LPS carry the same oligosaccharide moieties (PEA.sub.1.Math.Hex.sub.1.Math.Hep.sub.2.Math.HexNAc.sub.1.Math.Kdo.sub.2.Math.Gly.sub.1), with the exception of some LPS species, which lack a glycine or carry a second hexose residue (Hex) (Supplemental Table 2). Consequently, other differences observed between the LPS species, such as in the number of PEA and P groups, may be attributed to changes in the composition of the lipid A (Supplemental Table 2).

[0108] Analysis of the intact LPS from the ΔlpxL1 mutant revealed that the main ion peaks of the mass spectrum (3046.315, 3103.336, 3169.324 and 3226.342μ, FIG. 1B) had shifted compared to the 4 main ion signals of the LPS from the parent HB-1 (galE-) strain (FIG. 1A) by −182.165μ. This is in agreement with the lack of a dodecanoic acid (C12) (Δcalc.=−182.167μ) in the lipid A after deletion of the lpxl1 gene.

[0109] Comparative analysis of the mass spectrum of the LPS from the ΔlpxL2 mutant displayed ion peaks of 3023.367, 2966.348, 3185.419 and 3128.398 u (FIG. 1C), which are consistent with the loss of a C12 fatty acyl chain together with PPEA from the lipid A (Δcalc.=−385.142μ) in combination with non-stoichiometric substitution of the oligosaccharide with Gly (Δcalc.=57.021μ) or a second hexose (Δcalc.=162.053μ). This is in agreement with effective deletion of the lpxL2 gene. It is worthy to note that deletion of the lpxL2 gene not only led to the loss a C12 fatty acyl chain, as observed earlier upon deletion of the lpxL1 gene, but also resulted in the loss of a P and a PEA group from the lipid A.

[0110] The ion peaks in the mass spectrum of the LPS from the pagL mutant (3210.345, 3153.325, 3087.338 and 3030.318μ, FIG. 1D) were found to be shifted by −198.163μ from the 4 main ion peaks of the LPS from the parent HB-1 strain. This is in agreement with efficient removal of a 3-hydroxy-dodecanoic acid (C120H) (Δcalc.=−198.162μ) from the lipid A by the PagL enzyme. Nonetheless, display of minor ion peaks of 3408.505 and 3351.485 u (FIG. 1D) corresponding to unmodified hexa-acyl LPS species indicated that LPS 3-O-deacylation activity of the PagL enzyme could not fully exhaust the hexa-acyl lipid A substrate.

[0111] The 4 main ion signals in the mass spectrum of the LPS from the ΔlpxL1-pagL mutant (3028.180, 2971.160, 2905.173 and 2848.152μ, FIG. 1E) differed by −380.328μ from the 4 main ion signals of the LPS from the HB-1 strain, which is accordance with lack of a C12 and a C12OH in the lipid A of the ΔlpxL1-pagL mutant (Δcalc.=−380.329μ). The absence of ion signals corresponding to LPS carrying two C12 acyl chains indicates that the deletion of the lpxL1 gene resulted in complete removal of a single C12 from the lipid A (see Supplemental Table 1 for detailed LPS composition proposals). In contrast, minor ion signals of 3226.339 and 3169.319 u were present in the mass spectrum of the LPS from the ΔlpxL1-pagL mutant, which correspond to penta-acyl LPS species carrying two C120H acyl chains. This indicates that a low level of LPS molecules was not 3-O-deacylated by the PagL enzyme.

[0112] The mass spectrum of the LPS from the ΔlpxL2-pagL mutant showed an ion peak of 2825.206 u (FIG. 1F) that was shifted by −583.301 u from the ion signal of 3408.507μ of the mass spectrum of the LPS from the parent HB-1 strain (FIG. 1A). This fits the expected loss of a C12OH, a C12 and PPEA from the lipid A (Δcalc.=−583.304μ). Other ion signals of 2768.187, 2930.236 and 2987.257 u (FIG. 1F) are consistent with non-stoichiometric substitution of the oligosaccharide with Gly or a second Hex.

[0113] Comparison of the mass spectrum of the LPS from the ΔlpxL1-lpxP mutant grown at 30° C. (FIG. 1G) with that of the LPS from the ΔlpxL1 mutant (FIG. 1B) revealed that the LPS from the ΔlpxL1-lpxP mutant contained not only the main LPS species that were present in the LPS from the ΔlpxL1 mutant (3046.315, 3103.333, 3169.322 and 3226.340μ, FIG. 1G), corresponding to penta-acyl LPS lacking a C12, but also LPS species (3282.524, 3339.543, 3405.533 and 3462.553μ, FIG. 1G) that shifted in the spectrum to higher mass values by 236.211μ. This is in agreement with incorporation of a 9-hexadecenoic acid (C16:1) to the lipid A. Therefore, this preparation comprised a mixture of penta-acyl LPS that lacks a C12 and hexa-acyl LPS that lacks a C12 and additionally carry a C16:1.

[0114] The mass spectrum of the LPS from the ΔlpxL1-lpxP mutant cultured at 25° C. (FIG. 1H) showed ion signals corresponding to hexa-acyl LPS lacking a C12 and carrying additionally a C16:1(3282.526, 3339.546, 3405.535 and 3462.554, FIG. 1H), which were of a higher relative abundance as compared to the same signals in the spectrum of the LPS from the ΔlpxL1-lpxP mutant grown at 30° C. Furthermore, other ion peaks corresponding to hexa-acyl LPS carrying a C16:1 were displayed which arose from elongation of the oligosaccharide with a second Hex (3624.608μ) or the latter in combination with the loss of Gly substitution (3567.586μ) and the loss of a PEA group from the lipid A (3501.596μ) (FIG. 1H).

[0115] The ion peak of 3162.489 u in the mass spectrum of the LPS from the ΔlptA mutant (FIG. 1I) differed by −246.018 u from the ion signal of 3408.507 u of the mass spectrum of the LPS from the parent HB-1 strain (FIG. 1A). This points to the loss of two PEA groups from the lipid A (Δcalc.=−246.017μ). Other ion signals corresponded to LPS species that in addition to lacking PEA in the lipid A either lacked Gly in the oligosaccharide (3105.471), contained a second Hex in the oligosaccharide (3324.541) or contained a second Hex and lacked Gly in the oligosaccharide (3267.521μ) (FIG. 1I).

[0116] The mass spectrum of the LPS from the ΔlptA-ΔlpxL1 mutant displayed ion peaks of 2980.324, 2923.307, 3142.375 and 3085.354 u indicating the loss of 2PEA and a C12 from the lipid A (Δcalc.=−428.184μ) combined with non-stoichiometric substitution of the oligosaccharide with Gly or a second Hex (FIG. 1J). In addition, MS/MS spectra of the main lipid A fragment ion produced by in-source collision-induced dissociation of LPS were consistent with the presence of a P group at both the 1 and 4′positions of the lipid A (data not shown). Therefore, the activity of the LpxE enzyme consisted in removal of one of the three P groups present in lipid A producing bisphosphorylated lipid A species with a P group on each side of the diglucosamine backbone.

[0117] The main ion signals of the mass spectrum of the LPS from the ΔlptA-pagL mutant (2964.328 and 2907.311μ, FIG. 1K) are consistent with the loss of 2PEA and a C12OH from the lipid A (Δcalc.=−444.179μ) together with non-stoichiometric substitution of the oligosaccharide with Gly (Δcalc.=57.021μ). Minor ion peaks of 3105.468 and 3162.488 u were observed corresponding to hexa-acyl LPS species which lost only 2PEA from the lipid A, indicating a low level of incomplete LPS 3-O-deacylation by the PagL enzyme.

[0118] Finally, the mass spectrum of the LPS from the ΔlptA-lpxE mutant showed 2 main ion peaks of 3082.525 and 3025.508 u consistent with loss of 2PEA and P from the lipid A (Δcalc.=−325.983μ) in combination with non-stoichiometric substitution of the oligosaccharide with Gly (FIG. 1L).

2.3 TLR4 Stimulation by the LPS Mutant Strains

[0119] To determine the scope of TLR4 activation by the entire set of lipid A mutant structures, an initial screening was done using HEK-Blue humanTLR4 cells. These cells express human TLR4, MD-2, and CD14 and contain a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) dependent secreted embryonic alkaline phosphatase (SEAP) reporter gene. Stimulation of cells with serial dilutions of the different LPS mutants yielded a wide range of TLR4 activities (FIG. 2 and Table 5), with HB-1 inducing strongest TLR4 activation and ΔLpxL2 bacteria yielding lowest levels of activation. The other LPS mutants showed intermediate TLR4 stimulating activity (FIG. 2). A particularly notable result was that the absence of phosphoethanolamine in the ΔlptA strain resulted in reduced TLR4 activation both in the hexa-acylated wild type strain and the penta-acylated ΔlpxL1 and pagL backgrounds. Induction of LpxE in the ΔlptA strain showed similar TLR4 activation as ΔlptA strain, which was slightly less than the HB-1 wild type strain. This indicates that the reduction of three phosphates to two in the lipid A structure with one phosphate on each side of the diglucosamine backbone did not affect TLR4 signalling.

[0120] Expression of LpxP at 25° C. in combination with deletion of LpxL1 resulted in a heterogeneous hexa- and penta-acylated structure-LPS expressing strain with a slightly reduced TLR4 activating potential compared to the wild type bacteria. Cultivation of this strain at 30° C. resulted in less hexa-acylated lipid A and even slightly less TLR4 activity.

[0121] Surprisingly, when the ΔlpxL1 strain was combined with expression of PagL, reducing the penta-acylated lipid A structure to a tetra-acylated lipid A structure, an increase of TLR4 activity was obtained. This was unexpected as tetra-acylated lipid A structures typically acts as a TLR4 antagonists as reported for E. coli lipid Iva (7, 9, 28).

2.4 Human TLR4 Stimulation Using Purified Mutant LPS

[0122] We also purified LPS from all strains and used them to stimulate HEK-Blue TLR4 cells to confirm our initial findings with whole bacteria. Purified LPS generally yielded similar results as those obtained with intact bacteria although purified LPS, ΔlpxL1, ΔlptA-ΔlpxL1, ΔlpxL2 and ΔlpxL2-pagL showed almost no induction of TLR4 activity and were barely distinguishable from each other (FIG. 3), whereas the bacteria these variants displayed low but distinct TLR4 activities above the background. In addition, a higher concentration of purified penta-acylated pagL LPS was needed for activation of TLR4 than with all the hexa-acylated LPS derivatives, but with whole bacteria stimulation, a lower absorption density was necessary for the pagL strain to induce TLR4 activity than the other hexa-acylated mutant strain (FIG. 2+3). However, the maximum amount of alkaline phosphatase secretion was still lower for the pagL mutant strain compared to the hexa-acylated mutant strains. Of note, the three LPS mutants ΔlpxL1-pagL, pagL and ΔlptA-pagL had substantially reduced activating capabilities when compared to the wild type LPS, but still induced activation above the background level of unstimulated cells (FIG. 3).

2.5 Cytokine Induction by the Purified Mutant LPS

[0123] The cytokine induction profile of the modified LPS structures was investigated in the human monocytic cell line Mono Mac 6 (MM6). The concentration of secreted MyD88 dependent cytokines IL-6 (FIG. 4A and Table 6A) and IL-1β(FIG. 4BA and Table 6B) and TRIF dependent cytokines interferon gamma-induced protein 10 (IP-10) (FIG. 4C and Table 6C) and monocyte chemotactic protein-1 (MCP-1) (FIG. 4D and Table 6D) were determined after 20 h of stimulation with purified LPS (FIGS. 4E, F and G, and Table 7). The possible contribution of minor protein contamination in LPS samples to the observed responses was excluded as activation of a HEK-hTLR2 cell line by the LPS samples was negligible in the range of LPS concentrations tested (data not shown).

[0124] A wide variety of cytokine levels was determined from the different LPS structures, with the highest levels being produced by the HB-1 wild type hexa-acylated LPS and all other LPS ranging from close to wild type until virtually zero cytokine induction as seen for ΔlpxL2 LPS. Besides quantitative differences in cytokine induction, we also observed qualitative differences with LPS structures causing reduced levels of certain cytokines, but still capable of producing others. Some examples are pagL and ΔlptA-pagL LPS, which displayed a reduced capacity to induce the production of MyD88 dependent pro-inflammatory cytokines IL-6 and IL-1p only inducing 10% and 25% of the levels induced by wild-type LPS, respectively, but retained most of the ability to induce the secretion of TRIF dependent IP-10 (50%) and MCP-1 (90%). Interestingly, differences were observed between ΔlpxL1-lpxP grown at 30° C. and 25° C., with ΔlpxL1-lpxP grown at 30° C. producing 30-40% IL-6 and IL-1p and 60-85% of those cytokines at 25° C., whereas IP-10 and MCP-1 induction were similar. These results emphasize how LPS bioengineering can provide a wide range of agonists to fine-tune cytokine release.

3. Discussion

[0125] Although LPS has great potential as an adjuvant, adverse effects keep being a concern. Finding the optimal balance between adjuvant activity and minimal toxic effects requires the development of new LPS derivatives. Here we report a collection of novel meningococcal LPS structures inducing a broad range of TLR4 responses and differential cytokine patterns. These combinatorial bioengineered LPS mutants can be used as part of a whole cell vaccine, OMV vaccine or as purified LPS or lipid A molecule. OMVs of N. meningitidis are being actively investigated as potential vaccines and have been already approved for use in humans as a component of the Bexsero vaccine against serogroup B meningococcal disease (29,30). Attenuated ΔlpxL1 LPS is under investigation as constituent of meningococcal OMV vaccines and is a safe method to detoxify OMVs (16,31). In addition, in an immunization study purified ΔlpxL1 LPS retained similar adjuvant activity compared to wild type meningococcal LPS, but with reduced toxicity (15).

[0126] The modified LPS molecules LpxL1, LpxL2 and PagL all result in a reduced TLR4 activity compared to the parent strain (13,15). This was expected because they reduce the number of acyl chains in LPS from hexa to penta. Surprisingly, the expectation that tetra-acylated LPS is always less active than penta-acylated LPS is challenged by our results. Tetra-acylated lipid IVa of E. coli is a known antagonist of the human TLR4/MD-2 complex (7, 9, 28). Yet, we show that meningococcal tetra-acylatedΔlpxL1-pagL LPS is more active than the penta-acylated ΔlpxL1 LPS, whereas tetra-acylated ΔlpxL1-ΔlpxL2 LPS did not yield detectable activity (data not shown). Stimulation with ΔlpxL2-pagL whole bacteria that also carry a tetra-acylated LPS again increased TLR4/MD-2 activity compared to its penta-acylated ΔlpxL2 parent strain, although purified LPS from both the ΔlpxL2-pagL and ΔlpxL2 were inactive. Together these findings indicate that removal of C12OH from the 3′position by PagL in combination with deletion of a secondary acyl chain resulting in tetra-acylated lipid A yields a higher TLR4 activity compared to sole removal of the secondary acyl chain or both secondary acyl chains.

[0127] Interestingly, introduction of LpxP from E. coli into N. meningitidis conferred temperature-sensitive lipid A modification to N.meningitidis. Since conservation of temperature-sensitive gene expression signals is unlikely, this means that the enzyme itself is most active at lower temperatures. Selection of a temperature of 25 or 30° C. for culture of the ΔlpxL1-lpxP strain influenced the amount of hexa-acylated LPS species present in the mixture of penta- and hexa-acylated LPS produced by this mutant, with the lower temperature leading to the highest degree of substitution. The temperature sensitivity of the LpxP enzyme thus enables to prepare penta- and hexa-acylated LPS mixtures in a controlled manner. By selecting the time and/or temperature that the mutant strain is grown, it is feasible to increase or decrease the amount of hexa-acylated lipid A structure and thereby the TLR4 activity and cytokine profile. This provides a new approach of fine-tuning the immunological properties of meningococcal OMV vaccines.

[0128] In addition, we have obtained new insight in the specificity of the LpxE enzyme. Previously, the lpxE gene from Francisella tularensis or Francisella novicida expressed in E. coli was shown to be specific for the removal of the P group in the 1′position (32, 33). We have found that the lpxE homologue from B. bronchiseptica removed only one P group from the total of three present in the lipid A of N. meningitidis. MS/MS spectra of the lipid A from ΔlptA-lpxE mutants were consistent with the presence of a P group at both the 1 and 4′positions of the lipid A. In addition, removal of the P group was only seen in double ΔlptA-lpxE mutants, therefore only in the absence of PEA substitution of the lipid A. Thus, it is likely that the presence of PEA prevents lpxE from removing the P group. Most likely, the newly described LpxE enzyme is a pyrophosphatase, only catalysing hydrolysis between two phosphate groups. The absence of PEA in the lipid A through deletion of the lptA gene resulted in a reduced TLR4/MD-2 activity. This concurs with earlier observations by John et al. (34) that show a significant reduction of TNFα release by THP-1 cells upon stimulation with LptA lacking strains. Here we showed that reduction of the activity is even more apparent when stimulated with penta-acylated ΔlptA-pagL LPS or whole bacteria.

[0129] Interestingly, our results indicate that the absence of 2′ C12 fatty acyl chain by deletion of LpxL2 is accompanied by removal of a single P group and PEA group. This was previously not observed due to isolation of lipid A by an acid hydrolysis method before mass spectrometric analysis, which can result in the loss of P groups from the lipid A (15). In the present study, we used complete LPS molecules without introducing any deleterious chemical modifications for mass spectrometric analysis, giving us the possibility to observe new phosphorylation changes of the lipid A.

[0130] Several of the constructed attenuated LPS structures did not only need a higher concentration to induce TLR4 stimulation, but also did not yield the level of activation observed for the parent strain. This was most apparent for pagL LPS. The reason for this phenomenon is unclear, but could be due to instable dimerization of the LPS-TLR4-MD2 receptor complex at the cell surface but stable dimerization inside the cell, and/or to a less stable dimerization with high concentrations of the particular LPS. In addition, certain LPS species showed no activation at all and could potentially have antagonistic features, and might therefore serve as a TLR4 blocking drug. Indeed, meningococcal ΔlpxL1 and pagL penta-acylated LPS can block the TLR4 response when administered together with hexa-acylated wild type meningococcal LPS (13).

[0131] In the present study, we have used combinatorial bioengineering in meningococci to produce a range of LPS species with a broad array of TLR4 activity and cytokine profile. The application of these structures can be very broad, from inclusion into vaccines as adjuvants to their use in various forms of immunotherapy which have been described or suggested for LPS, such as cancer therapy, Alzheimer's disease or generalized immune stimulation to prevent diverse infections (3, 35-37).

TABLE-US-00004 TABLE 4 Multiple sequence alignment of the LpxP proteins. Sequences were aligned using ClustalW (2.1) (http://clustalw.ddbj.nig.ac.jp/) using default settings and the Gonnet protein weight matrix. Hyphens indicate gaps introduced for optimal alignment. Absolutely conserved residues are marked with asterisks. Colons and dots indicate strongly and weakly conserved residues, respectively. Sequences are from Escherichia coli (Genbank AAB66658), Citrobacter youngae (Genbank EFE09970), Haemophilus aegyptius (Genbank EGF15820), Klebsiella sp. (Genbank EFD82732), Salmonella enterica subsp. enterica serovar Typhimurium (Genbank CBW18471), Serratia plymuthica (Genbank EKF62147) and Shigella flexneri (Genbank ADA74790). AAB66658 MFPQ--CKFSREFLHPRYWLTWFGLGVLWLWVQLPYPVLCFLGTRIGAMARPFLK--RRE 56 ADA74790 MFPQ--CKFSREFLHPRYWLTWFGLGVLWLWVQLPYPVLCFLGTRIGAMARPFLK--RRE 56 CBW18471 MFPQ--SKFSRAFLHPRYWLTWFGVGILWLLVQLPYPVLRFLGTRTGKLARPFLK--RRE 56 EFE09970 MFPQ--CKFSRAFLHPRYWLTWFGVGVLWLLVQLPYPLLCFLGTRTGTLARPFLK--RRE 56 EFD82732 --MA--CVFNKQLLHPRNWLTWFGLGILWLIVQLPYPLLHFIGTSAGRLSRRFLK--RRE 54 EKF62147 MKRP--QEFRSALLHPRYWFTWFGLAILFLLVQLPYPLLHKLGVWMGRTSMRFLK--RRV 56 EGF15820 MKNEKLPQFQPHFLVPKYWLFWLGVAIWRSILCLPYPILRHIGHGLGWLFSHLNEGKRRA 60         *   :* *: *: *:*:.:    : ****:*  :*   *     : :  ** AAB66658 SIARKNLELCFPQHSAEEREKMIAENFRSLGMALVETGMAWFWPDSRVRKWFDVEGLDNL 116 ADA74790 SIARKNLELCFPQHSAEEREKMIAENFRSLGMALVETGMAWFWPDSRVRKWFDVEGLDNL 116 CBW18471 SIAQKNIELCFPTLSREEREKLIAENFHSLGMALLETGMAWFWPDSRVRKWFDVDGLDNL 116 EFE09970 SIARKNLELCFPNLSQEERDKLVDENFRSLGMGLLETGMAWFWPDRRVRKWFDVEGLDNL 116 EFD82732 HIARRNIELCFPDMSPAARETLIDQNFMSLGMGLIETGMAWFWSDERVKKWFDVEGFANL 114 EKF62147 AITRRNLELCFPDMDEAQRERKVIGNFESLGMGLLETGMAWFWSDKRVKRWFNVSGINHL 116 EGF15820 AIARRNLELCFPYMPENERETILQENLRSVGMAIIETGMAWFWSDSRIKKWSKVEGLHYL 120  *:::*:*****      *:  :  *: *:**.::********.* *:::* .*.*:  * AAB66658 KRAQMQNRGVMVVGVHFMSLELGGRVMGLCQPMMATYRPHNNQLMEWVQTRGRMRSNKAM 176 ADA74790 KRAQMQNRGVMVVGVHFMSLELGGRVMGLCQPMMATYRPHNNQLMEWVQTRGRMRSNKAM 176 CBW18471 TRAQAQNRGVMVVGVHFMSLELGGRVMGLCQPMMATYRPHNNPLMEWVQTRGRMRSNKAM 176 EFE09970 QRAQIEGRGVMVVGVHFMSLELGGRVMGLCQPTMATYRPHNNKLMEWIQTRGRMRSNKAM 176 EFD82732 NHALSGGKGVMVVGVHFMSLELGGRAMGLCRPMMATYRPHNSPLMEWVQTRGRLRSNKAM 174 EKF62147 KMAQQNERGVLVIGVHFMSLELGGRAMGLCQPMMAMYRPHNNKAMEWAQTKGRMRSNKAM 176 EGF15820 KENQKD--GIVLVGVHFLTLELGARIIGLHHPGIGVYRPNDNPLLDWLQTQGRLRSNKDM 178         *::::****::****.* :** :* :. ***::.  ::* **:**:**** * AAB66658 IGRNNLRGIVGALKKGEAVWFAPDQDYGRKGSSFAPFFAVENVATTNGTYVLSRLSG-AA 235 ADA74790 IGRNNLRGIVGALKKGEAVWFAPDQDYGRKGSSFAPFFAVENVATTNGTYVLSRLSG-AA 235 CBW18471 IGRNNLRGIVGALKKGEAVWFAPDQDYGPKGSSFAPFFAVENVATTNGTYVLSRLSG-AA 235 EFE09970 IGRNNLRGIVGALKKGEAVWFAPDQDYGRKGSSFAPFFAVKDVATTNGTYVLSRLSG-AA 235 EFD82732 IDRNNLTGLVHALKSGEAVWFAPDQDYGPKGSVFAPFFSVPQAATTNGTYVLSRLSG-AK 233 EKF62147 LDRKDLRGMVHALKRGEAVWFAPDQDYGPRGSVFAPLFAVDQAATTSGTFMLARMAK-PA 235 EGF15820 FDRKDLRGMIKALRHEETIWYAPDHDYGRKNAVFAPFFAVPDACTTTGSYYLLKSSQNSK 238 :.*::* *:: **:  *::*:***:*** :.: ***:*:* :..**.*:: * : :  . AAB66658 MLTVTMVRKADYSGYRLFITPEMEGYPTDENQAAA-YMNKIIEKEIMRAPEQYLWIHRRF 294 ADA74790 MLTVTMVRKADYSGYRLFITPEMEGYPTDENQAAA-YMNKIIEKEIMRAPEQYLWIHRRF 294 CBW18471 MLTVTMVRKSDNSGYRLYITPEMEGYPADENQAAA-YMNKIIEKEIMRAPEQYLWIHRRF 294 EFE09970 MLTVTMVRKADNSGYRLFITPQMEGYPADESQAAA-YMNKIIEKEIMRAPEQYLWIHRRF 294 EFD82732 MLSISMVRKLDRQGYSLHISEVMNDYPGEDKQIAAGYINKVIEREILRAPEQYLWVHRRF 293 EKF62147 LVPVVLIRREKGRGYDLLIQPALEDYPIGDELAAAAYMNKVVEKEIMRAPEQYMWLHRRF 295 EGF15820 VIPFAPLRNKDGSGYTVSISAPVDFTDLQDETAIATRMNQIVEKEIMKGISQYMWLHRRF 298 ::..  :*. .  ** : *   ::     :.   *  :*:::*:**::. .**:*:**** AAB66658 KTRP-VGESSLYI 306 ADA74790 KTRP-VGESSLYI 306 C17W18471 KTRP-LGEASLYI 306 EFE09970 KTRP-MGEASLYI 306 EFD82732 KTRP-LGEPSVY- 304 EKF62147 KTRP-IGAPSLY- 306 EGF15820 KTRPDEKTPSLYD 311 ****    .*:*

TABLE-US-00005 TABLE 5 TLR4 activation by N. meningitidis strains (the same data are graphically presented in FIG. 2A). HEK-blue hTLR4 cells were stimulated with 5-fold serial dilutions at A600 nm (y-axis) of heat-inactivated N. meningitidis for 20 h. TLR4 activation was measured by detection of secreted alkaline phosphatase at A649 nm. Data are expressed as mean values of three independent experiments. ΔL1- ΔL1- ΔL1- ΔLa- A600 nm HB-1 ΔL1 ΔL2 P ΔL1-P ΔL2-P Lp37 Lp30 Lp25 ΔLa ΔL1 Δla-P ΔLa-E 0.01 1.102 0.318 0.124 0.715 0.596 0.277 0.534 1.07 1.011 0.874 0.157 0.501 0.911 0.002 1.083 0.247 0.092 0.692 0.569 0.199 0.45 0.967 0.964 0.855 0.098 0.469 0.872 0.0004 1.093 0.187 0.096 0.703 0.536 0.172 0.340 0.925 0.974 0.856 0.090 0.431 0.915 0.00008 1.031 0.124 0.100 0.723 0.387 0.115 0.217 0.607 0.687 0.836 0.087 0.354 0.840 1.6E−05 0.959 0.089 0.094 0.669 0.164 0.111 0.146 0.301 0.344 0.553 0.088 0.156 0.466 3.2E−06 0.551 0.101 0.092 0.377 0.100 0.094 0.137 0.189 0.162 0.219 0.083 0.107 0.174 6.4E−07 0.239 0.089 0.100 0.164 0.104 0.108 0.135 0.167 0.116 0.098 0.079 0.081 0.106 1.3E−07 0.141 0.095 0.116 0.125 0.101 0.110 0.138 0.169 0.102 0.087 0.094 0.089 0.090 2.6E−08 0.131 0.101 0.097 0.115 0.099 0.104 0.141 0.171 0.103 0.089 0.087 0.091 0.092 5.1E−09 0.134 0.095 0.108 0.108 0.098 0.114 0.158 0.188 0.101 0.088 0.085 0.095 0.094   1E−09 0.151 0.107 0.119 0.120 0.104 0.120 0.165 0.205 0.133 0.098 0.090 0.111 0.112

TABLE-US-00006 TABLE 6 Cytokine release of MM6 cells stimulated with purified LPS (the same data are graphically presented in FIG. 4). MM6 cells were incubated with 10-fold serial dilution of different LPS mutants for 20 h. IL-6, IP-10, IL-1β, MCP-1production was measured by ELISA. Data shown are depicted as the mean values in pg/mL of two independent experiments. LPS (ng/mL) HB-1 ΔL1 ΔL2 P ΔL1-P ΔL2-P ΔL1-Lp30 ΔL1-Lp25 ΔLa ΔLa-ΔL1 ΔLa-P ΔLa-E A: IL-6 (pg/mL) 5 2418.74 8.19 7.85 298.35 16.78 3.40 519.86 1490.81 1471.22 7.96 64.14 1868.77 0.5 1563.59 1.01 3.02 185.57 8.53 1.13 320.98 877.57 819.39 3.90 50.82 1054.23 0.05 368.77 0.00 1.51 7.54 0.28 2.59 51.98 185.12 159.19 2.52 11.49 287.74 0.005 23.15 0.00 1.88 0.00 0.00 0.00 2.35 15.92 8.49 3.29 0.39 18.04 0.0005 0.00 0.00 0.73 1.45 0.00 0.28 0.01 1.49 1.71 4.15 0.08 1.35 B: IP-10 (pg/mL) 5 2285.04 82.21 62.66 1325.52 322.33 104.11 1615.79 1933.60 2146.18 153.76 1247.00 2286.09 0.5 2039.96 32.05 40.90 1100.20 260.54 35.18 1594.91 1696.40 1904.13 49.04 827.49 2196.92 0.05 1378.14 2.32 12.51 231.93 30.34 75.39 784.55 1162.30 1142.07 12.36 296.77 1688.99 0.005 449.26 0.00 0.00 4.43 17.83 0.00 104.71 373.72 239.53 19.44 24.38 511.23 0.0005 55.89 0.00 0.00 0.00 0.00 0.00 20.82 42.53 34.85 12.55 17.63 64.73 C: IL-1β (pg/mL) 5 883.53 26.80 26.06 109.46 31.74 21.90 207.76 666.32 516.85 28.80 46.79 880.08 0.5 430.02 19.36 26.28 58.08 23.39 21.29 111.23 312.46 213.05 27.70 40.54 394.26 0.05 90.35 17.73 18.01 18.87 13.16 14.47 34.21 73.87 63.95 23.69 22.59 100.26 0.005 15.50 10.94 10.45 14.46 9.82 14.37 26.28 28.38 31.30 27.03 22.73 30.80 0.0005 13.46 11.31 11.18 15.06 6.98 11.68 17.93 22.72 20.74 28.69 23.05 27.98 D: MCP-1 (pg/mL) 5 5248.05 1587.37 1340.99 4983.60 2780.68 1416.04 5248.05 5248.05 5248.05 1442.22 4251.98 5127.77 0.5 5248.05 1309.56 1156.87 4127.69 1449.70 1085.53 5248.05 5248.05 5248.05 934.29 3771.32 5020.45 0.05 5248.05 1172.01 1085.39 1370.45 1053.58 1046.54 4864.54 5158.56 4237.64 777.19 1470.83 4211.41 0.005 3338.36 1287.71 1166.78 942.49 1323.53 1357.65 1459.20 3657.48 2654.60 869.76 1651.58 2457.90 0.0005 1807.16 1201.89 1038.39 901.63 1122.21 833.56 1763.86 1466.27 1172.02 923.73 1191.91 1176.04

TABLE-US-00007 TABLE 7 Cytokine release in percentages of MM6 cells stimulated with purified LPS (the same data are graphically presented in FIG. 4). MM6 cells were stimulated with 5 ng/ml LPS. Data are expressed as mean values of two independent experiments. IL-6 (%) SEM IL-1b (%) SEM IP-10 (%) SEM MCP-1 (%) SEM IL-10 (%) SEM HB-1 100.00 4.07 100.00 6.08 100.00 7.68 100.00 0.00 100.00 19.63 ΔL1 0.34 0.20 3.03 0.72 3.60 0.88 30.25 1.38 12.26 7.49 ΔL2 0.32 0.20 2.95 0.69 2.74 0.35 25.55 2.30 11.69 6.77 P 12.33 0.75 12.39 0.40 58.01 5.08 94.96 5.04 41.61 14.45 ΔL1-P 0.69 0.34 3.59 0.86 14.11 1.89 52.99 16.31 15.41 8.41 ΔL2-P 0.14 0.08 2.48 0.96 4.56 0.60 26.98 1.83 11.39 6.58 ΔL1-Lp30 21.49 2.86 23.51 5.58 70.71 11.46 100.00 0.00 48.97 8.55 ΔL1-Lp25 61.64 6.63 75.42 9.59 84.62 9.85 100.00 0.00 79.72 9.28 ΔLa 60.83 3.58 58.50 8.23 93.92 13.41 100.00 0.00 79.33 12.54 ΔLa-ΔL1 0.33 0.09 3.26 0.92 6.73 1.41 27.48 1.41 12.74 7.48 ΔLa-P 2.65 0.28 5.30 0.46 54.57 5.77 81.02 11.52 22.94 11.66 ΔLa-E 77.26 6.43 99.61 9.70 100.05 13.99 97.71 2.29 83.23 13.21 No LPS 0.12 0.05 3.62 1.01 2.41 0.82 22.94 3.23 10.69 6.27

TABLE-US-00008 SUPPLEMENTAL TABLE 1 Composition of the main ion peaks observed in charge-deconvoluted ESI-FT mass spectra of intact LOS from twelve mutants of N. meningitidis (see FIG. 1). Calcu- Devi- Measured Proposed LOS composition lated ation Bacteria mass (u) Oligosaccharide Lipid A mass (u) (ppm) HB-1 3408.507 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3408.514 2.2 3351.488 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3351.493 1.5 3285.501 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3285.506 1.5 3228.480 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3228.484 1.4 ΔlpxL1 3226.342 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3226.347 1.7 3169.324 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3169.326 0.6 3103.336 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3103.339 0.9 3046.315 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3046.317 0.8 3388.394 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3388.400 1.8 3331.373 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3331.379 1.7 ΔlpxL2 3023.367 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3023.373 1.8 2966.348 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2966.351 1.0 3185.419 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3185.425 2.0 3128.398 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3128.404 1.9 2843.340 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2843.343 0.9 2720.331 Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2720.334 1.1 pagL 3210.345 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3210.352 2.3 3232.326* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3232.335 2.8 3153.325 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3153.331 1.9 3175.306* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3175.313 2.4 3087.338* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3087.344 1.9 3109.320* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3109.326 2.1 3030.318 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3030.322 1.5 3052.298* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3052.305 2.3 2971.161 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2971.164 1.0 2950.352 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2950.356 1.4 2848.152 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2848.155 1.2 3408.505 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3408.514 2.8 3351.485 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3351.493 2.4 3372.396 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3372.405 2.8 3315.376 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3315.384 2.4 ΔlpxL1- 3028.180 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 3028.185 1.8 pagL 2971.160 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2971.164 1.3 2905.173 PEA.sub.1•Hex•.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2905.177 1.3 2848.152 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2848.155 1.2 3226.339 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3226.347 2.6 3169.319 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3169.326 2.2 3190.232 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 3190.238 2.0 3133.210 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 3133.217 2.2 3103.331 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3103.339 2.5 ΔlpxL2- 2825.206 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2825.211 1.6 pagL 2768.187 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2768.189 0.8 2987.257 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Glyl P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2987.263 2.1 2930.236 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2930.242 2.0 2966.344 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2966.351 2.4 2548.127 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.1 P.sub.2•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2548.131 1.5 2645.178 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2645.181 1.0 2720.331 Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2720.334 1.1 ΔlpxL1- 3046.315 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3046.317 0.8 lpxP 3103.333 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3103.339 1.9 30° C. 3169.322 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3169.326 1.2 3226.340 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3226.347 2.3 3208.364 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3208.370 1.9 3265.382 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3265.392 3.0 3331.372 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3331.379 2.0 3388.391 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3388.400 2.7 3282.524 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3282.531 2.3 3339.543 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3339.553 3.0 3405.533 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3405.540 2.0 3462.553 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3462.561 2.4 3444.574 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3444.584 3.0 3567.585 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3567.593 2.2 3624.606 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3624.614 2.3 ΔlpxL1- 3046.314 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3046.317 1.1 lpxP 3103.333 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3103.339 1.9 25° C. 3169.321 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3169.326 1.6 3226.341 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3226.347 2.0 3208.366 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3208.370 1.3 3265.386 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3265.392 1.7 3331.373 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3331.379 1.7 3388.393 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3388.400 2.1 3254.494 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 —C2H4 3254.500 1.9 3282.526 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3282.531 1.6 3311.516 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 —C2H4 3311.522 1.7 3339.546 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3339.553 2.1 3377.502 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 —C2H4 3377.509 2.0 3405.535 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3405.540 1.4 3434.522 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 —C2H4 3434.530 2.4 3462.554 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3462.561 2.1 3444.575 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3444.584 2.7 3501.596 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3501.606 2.8 3567.586 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3567.593 1.9 3624.608 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•PEA.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 3624.614 1.7 ΔlptA 3162.489 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3162.497 2.7 3184.471* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Glyl P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3184.480 2.8 3105.471 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3105.476 1.6 3127.452* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3127.458 2.0 3025.506 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3025.510 1.2 3324.541 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3324.550 2.8 3267.521 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3267.529 2.4 ΔlptA- 2980.324 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2980.330 2.1 ΔlpxL1 2923.307 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 2923.309 0.6 3142.375 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3142.383 2.6 3085.354 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 3085.362 2.5 ΔlptA- 2964.328 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2964.335 2.5 pagL 2986.309* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2986.318 3.0 2907.311 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2907.314 1.0 2929.290* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2929.296 2.2 3069.359 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3069.367 2.5 3105.468 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3105.476 2.6 3126.379 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 3126.388 3.0 3162.488 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3162.497 3.0 2725.144 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.3•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2725.147 1.1 2782.165 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.3•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 2782.168 1.2 2827.344 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2827.348 1.3 2687.254 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.1 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 2687.256 0.6 ΔlptA- 3082.525 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3082.531 2.0 lpxE 3104.507* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3104.514 2.1 3025.508 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3025.510 0.5 3047.488* PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3047.492 1.3 3069.468** PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3069.475 2.1 3244.576 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3244.584 2.4 3187.556 PEA.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 3187.562 2.0 2805.449 PEA.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.1 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 2805.451 0.8 *Monosodium adduct. **Disodium adduct. Abbreviations: Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Hep, L-glycero-D-manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine; Gly, glycine; PEA, phosphoethanolamine; P, phosphate; C12OH, 3-hydroxy-dodecanoic acid; C14OH, 3-hydroxy-tetradecanoic acid; C12, dodecanoic acid; C16:1, 9-hexadecenoic acid

TABLE-US-00009 TABLE 2 Proposed compositions for charge-deconvoluted fragment ion peaks obtained by in-source collision-induced dissociation ESI-FT MS of LOS. Fragment ion Measured Proposed LOS composition Calculated Deviation Bacteria type.sup.a) mass (u) Oligosaccharide Lipid A mass (u) (ppm) HB-1 B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 B-Kdo-CO2 1105.355 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 2.2 B-Kdo-CO2 1048.334 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 1.9 Y 1916.098 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 1916.100 1.2 Y 2039.106 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 2039.109 1.4 ΔlpxL1 B 1369.405 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 0.4 B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 Y 1733.933 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1733.933 0.2 Y 1856.942 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1856.942 0.1 ΔlpxL2 B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.409 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.0 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1530.957 P.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1530.958 0.9 Y 1653.965 P.sub.2•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1653.967 1.2 pagL B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 B 1312.381 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1312.384 2.4 B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1717.937 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 1717.938 0.8 Y 1840.946 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 1840.947 0.5 ΔlpxL1- B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 pagL B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.409 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.0 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1535.77 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 1535.771 0.8 Y 1658.779 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 1658.780 0.5 Y 1856.943 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1856.942 0.6 Y 1733.933 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1733.933 0.2 ΔlpxL2- B-Kdo-CO2 1105.355 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 2.2 pagL B-Kdo-CO2 1048.334 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 1.9 B-Kdo-CO2 1267.408 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.8 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1455.802 P.sub.2•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.1•C14OH.sub.2 1455.805 2.0 Y 1653.964 P.sub.2•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1653.967 1.8 Y 1530.955 P.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1530.958 2.2 ΔlpxL1- LpxP B 1369.403 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.9 30° C. B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.408 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.8 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1856.941 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1856.942 0.4 Y 1733.932 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1733.933 0.7 Y-P 1653.965 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1653.967 1.2 Y-2P-H2O 1555.988 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1555.990 1.3 Y 2093.153 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 2093.156 1.4 Y 1970.147 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 1970.147 0.2 Y-P 1890.182 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 1890.181 0.5 Y-2P-H2O 1792.202 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 1792.204 1.2 ΔlpxL1- B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 LpxP B 1312.379 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1312.384 3.9 25° C. B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.409 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.0 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1856.941 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1856.942 0.4 Y 1733.932 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1733.933 0.7 Y-P 1653.965 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1653.967 1.2 Y-2P-H2O 1555.988 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2 1555.990 1.3 Y 2093.152 P.sub.3•PE.sub.2•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 2093.156 1.8 Y 1970.147 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 1970.147 0.2 Y-P 1890.18 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 1890.181 0.5 Y-2P-H2O 1792.203 P.sub.3•PE.sub.1•HexN.sub.2•C12.sub.1•C12OH.sub.2•C14OH.sub.2•C16:1.sub.1 1792.204 0.6 ΔlptA B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 B 1312.38 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1312.384 3.1 B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.408 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.8 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1793.09 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 1793.092 1.0 Y 1713.123 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 1713.125 1.5 ΔlptA- B 1369.405 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 0.4 ΔlpxL1 B 1312.38 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1312.384 3.1 B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.409 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.0 B-Kdo-CO2 1210.388 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 0.7 Y 1610.923 P3•HexN.sub.2•C121.sub.2•C12OH.sub.2•C14OH.sub.2 1610.925 1.1 ΔlptA- B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 pagL B 1312.382 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1312.384 1.6 B-Kdo-CO2 1105.356 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 1.3 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1267.409 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1267.410 1.0 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1594.928 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 1594.930 1.2 Y 1514.961 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.1•C14OH.sub.2 1514.964 1.7 Y 1793.092 P.sub.3•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 1793.092 0.1 Y 1713.125 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 1713.125 0.3 ΔlptA- B 1369.404 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1369.406 1.1 LpxE B 1312.382 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1312.384 1.6 B 1474.436 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1474.437 0.6 B-Kdo-CO2 1105.355 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2•Gly.sub.1 1105.357 2.2 B-Kdo-CO2 1048.335 PE.sub.1•Hex.sub.1•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1048.336 0.9 B-Kdo-CO2 1210.387 PE.sub.1•Hex.sub.2•Hep.sub.2•HexNAc.sub.1•Kdo.sub.2 1210.389 1.5 Y 1713.124 P.sub.2•HexN.sub.2•C12.sub.2•C12OH.sub.2•C14OH.sub.2 1713.125 0.9 In-source collision-induced dissociation of LOS produced B- and Y-type fragment ions corresponding to oligosaccharide and lipid A domains due to the rupture of the glycosidic bond between Kdo and lipid A. Fragment ions are assigned according to the nomenclature of Domon and Costello (24). Mass numbers given refer to monoisotopic masses of the neutral molecules. Abbreviations: Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Hep, L-glycero-D-manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine; Gly, glycine; PEA, phosphoethanolamine; P, phosphate; C12OH, 3-5 hydroxy-dodecanoic acid; C14OH, 3-hydroxy-tetradecanoic acid; C12, dodecanoic acid; C16:1, 9-hexadece

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