MAb WN1 222-5 in context

Monoclonal Antibodies to Endotoxin Core Segregate into Families of Specificity and Cross-Reactivity to Lipopolysaccharides in ELISA by Correlation Cluster Analysis.

G. Robin Barclay et al.

e-mail: r.barclay@virgin.net

Abstract

A series of 291 mouse monoclonal anti-endotoxin antibodies predominantly with specificity for epitopes in the LPS cores in various endotoxins were assayed in ELISA to a large panel (66) of rough and smooth LPS and lipid A from a variety of sources and species. The resultant data was sorted according to relationships between MAbs and between LPS antigens, so that similar MAbs congregated together and similar LPS congregated together. The MAbs segregated into 24 major family groups. The LPS segregated into clusters which reflected structural similarities. Some MAb families displayed reactivity for either Re, Re-Rd, or Rc chemotype LPS and did not react with more complete rough or smooth LPS. Some MAb families were specific for one or more core types from E.coli and Salmonella: within this category some MAbs apparently retained core-type specificity in rough (Ra) and smooth LPS, while others showed core-type cross-reactivities on R-LPS but more restricted specificity on S-LPS. Other MAbs families were identified with specificities for Rc or Re or lipid A minimal binding structures which showed wide cross-reactivity with larger LPS including S-LPS from different species and strains of Gram-negative bacteria. Some of these have potent endotoxin neutralising activity in vitro and in vivo.

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Abbreviations

BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; KDO, 3-deoxy-D-mannooctulosonic acid; LPS, lipopolysaccharide; MAb, monoclonal antibody; R-LPS, rough LPS; S-LPS, smooth-LPS;

Introduction

The endotoxins of Gram-negative bacteria are lipopolysaccharide (LPS) molecules with three distinct domains referred to as lipid A, core oligosaccharide and O-polysaccharide. The lipid A and core oligosaccharide comprise the endotoxin core and are relatively conserved among different Gram-negative bacterial species. The O-polysaccharides show wide structural diversity and give rise to the O-specificity of different strain and species serotypes. Lipid A is the toxic moiety of endotoxin, and is covalently linked to core oligosaccharide in all LPS [1]. Rough (R) bacteria lack the O-polysaccharide of their smooth (S) strain bacteria counterparts, and different rough mutant bacteria have been isolated expressing a range of incomplete core oligosaccharide structures ranging from complete core (Ra) to deep-rough (Re) mutants expressing only lipid A linked to inner-core KDO residues [2,3]. R-LPS from these mutants has been invaluable in analysis of specificity and cross-reactivity of anti-core antibodies.

Cross-reactive LPS-core specific antibodies are found in all healthy adults [4,5,6]. These antibodies approach adult levels by one year old in infants [7]; they become acutely depressed in life-threatening Gram-negative sepsis [8,9,10]; and they are elevated following immunogenic natural exposure to endotoxin [11,12]. The hypothesis that such antibodies provide an important host defence [13] has stimulated the pursuit of polyclonal [14,15] and monoclonal [16,17] antibodies with these cross-reactive endotoxin-neutralising properties for immunotherapeutic use, with disappointing results [15,18]. Many of these studies have employed lipid A or R-LPS (or R-mutant bacteria) as immunogens, in an effort to employ only the most structurally conserved regions of LPS to elicit cross-reactive antibodies and avoid elicitation of serovar specific antibodies. However, the LPS core region has proved immunochemically more complex than was hypothesised, and the MAbs which promised new therapeutic widely reactive anti-endotoxin reagents have shown, in general, exquisite but restricted specificity ranges [19].

Rough bacteria are serum-sensitive. Only complete-core (Ra) R-LPS, co-expressed with S-LPS on smooth bacteria, occurs commonly with S-LPS in the host, contributing to pathogenesis and acting as immunogens eliciting natural polyclonal anti-endotoxin antibodies. Large numbers of MAbs have been reported which appear to react predominantly with lipid-A-distal hydrophilic moieties which, in many cases, represents the exposed end of the oligosaccharide chain from the particular R-LPS chemotype employed for immunisation [19]. In incomplete-core R-LPS and lipid A, such moieties represent breaks in the complete core structure, and are therefore novel antigens when compared to the natural LPS primary structures seen by the host. Such MAbs fail to react convincingly with natural, clinically important complete LPS and show little or no endotoxin neutralising activity with these LPS. It is also probable that the incomplete core R-LPS express different secondary and tertiary structures from complete core R-LPS and the core of S-LPS, affecting shape and aggregation properties which may contribute to antigenic structures [20].

We have succeeded in producing widely cross-reactive, LPS-core-specific, endotoxin-neutralising MAbs by employing immunisation procedures which mimic natural exposure to endotoxins [21]. We predominantly used complete core (Ra chemotype) bacteria or LPS, and cyclical immunisations were carried out to expose animals to different LPS-core serovars, in an attempt to boost cross-reactive immunity to the common structures shared by the different immunogens. Primary hybridoma screens employed multiple ELISA analyses with different individual LPS and LPS cocktails, to preferentially select multiply-reactive MAbs. The study was carried out as a collaborative exercise in two geographically distant centres in Edinburgh and Basle, over four years, from which 291 MAb-secreting hybridoma lines were retained, some of whose properties have been reported [21, 22]. Although it has been possible to examine the specificity of some of these MAbs in detail, a comprehensive and systematic analysis of this bank of anti-LPS MAbs has proved intractable because of the number of MAbs involved, and because of the number of LPS antigens against which they were tested in ELISA, for which only minimal or inferred structural information was available concurrently. We now report a method we have devised for evaluating relationships in a complex ELISA system, which simultaneously permits systematic analysis of MAb:MAb interrelationships and antigen:antigen (LPS:LPS) serological interrelationships. The method is based on correlation between ELISA reactivities, and congregates MAbs (or LPS) of similar reactivities together into serologically related families without any prior knowledge of MAb specificity or presumption of LPS structure. The reaction patterns of MAb families and LPS families can then be retrospectively analysed systematically, for assignation of presumptive MAb family LPS specificity, and assignation of presumptive LPS family serological properties with reference to published structure information.

Methods

Bacterial strains, LPS and lipid A.

The 66 LPS used in ELISA are shown in Table 1. Where indicated, including lipid A, these were purchased from List Biologicals, CA, USA (List) or Sigma Chemicals, Poole, England (Sigma). The remaining LPS were produced from the respective bacterial strains in the Department of Medical Microbiology, University of Edinburgh, as previously reported [5]. Smooth E.coli of known O-serotype were obtained from Alan S Cross (Walter Reed Army Institute of Research, Washington DC, USA) and included 2 smooth variants of E.coli O18 (Bort) comprised of the original K1 (K1+) encapsulated strain and its unencapsulated (K1-) mutant: a complete core rough mutant of E.coli O18 K1+ (Ra ex O18) was also included, as previously described [21]. Examples of rough E.coli core type strains R1 (HF4704), R2 (EH100), R3 (F673) and R4 (F2513), marked (k), were obtained from Nils Carlin (Karolinska Institute, Stockholm, Sweden), as was the rough E.coli C62. Further examples of rough E.coli core type strains R1 (F470), R2 (F576), R3 (F653) R4 (F2513), K-12 (W3100) and Re (F515), marked (b), were obtained from Helmut Bräde (Forschungsinstitut Borstel, Germany), as was smooth E.coli O4. Salmonella typhimurium rough mutants Ra (1542), Rb (119), Rc (878), Rd (1032) and Re (1102) were obtained from Ian Sutherland (Dept of Microbiology, University of Edinburgh). Pseudomonas aeruginosa rough (Rc) (PAC605) was obtained from Pauline Meadow (University College, London). Klebsiella aerogenes Rb (M10b) was isolated by Ian Poxton.

Heat-killed or formalin-killed bacteria used in immunisations in Edinburgh and Basle were prepared in the Department of Medical Microbiology, University of Edinburgh.

Monoclonal Antibodies.

Monoclonal antibodies were generated by standard methods. The particular details of methods used in the Basle [21] and Edinburgh [23] laboratories have been previously described. A variety of immunisation procedures were employed. In most cases heat-killed bacteria were used. In many cases cyclical immunisations were carried out (see introduction) with complete-core (Ra) bacteria of different core structure. In other cases, different R-LPS variants were used (Ra to Re), and in some cases R-LPS from different species were used, in cyclical immunisations. Other immunisations, e.g. with purified R-LPS or with lipid A coated on mouse red cells, were carried out. In general, mice received immunisations at 4-week intervals, and tail-bleeds were taken at weekly intervals to investigate development of multiple LPS specificity by ELISA to selected LPS panels. Final immunisations of selected immune mice prior to spleen recovery varied from fusion to fusion, but were generally cocktails of the different immunising bacteria when cyclical immunisations were employed.

The ELISAs for hybridoma screening have been previously described [21, 23]. The Basle ELISA employed purified LPS or killed bacteria on PVC microtitre plates [21], while the Edinburgh ELISA employed polymyxin B-complexed purified LPS on polystyrene microtitre plates [4,5]. Primary screens of hybridoma supernatants employed multiple analyses on selected single or cocktail LPS, which varied from fusion to fusion. In most cases only hybridomas whose supernatants showed multiple or unexpected LPS reactivity patterns were retained for cloning and extended specificity analyses. Retained clones were grown beyond maximum supportable cell density for harvesting supernatants for detailed ELISA analysis. Samples of supernatants from all 291 retained cloned hybridoma cell lines were collected in Edinburgh for standardised ELISA assay, and stored at -40ºC until tested.

ELISA.

The standardised ELISA used to obtain the results presented here employed purified LPS complexed with polymyxin B and coated on 8-well polystyrene microtitre plate strips as previously described [5]. Batches of strips were coated with LPS-polymyxin, blocked with BSA, and stored at -40ºC until required. Strips were assembled in microtitre strip-plate frames to give 11 different LPS antigens in each frame: the last (12th) strip in each frame was coated with BSA only and was used as a negative control. The full ELISA required a set of 6 plates. Any empty spaces in the last (6th) frame were filled with uncoated strips. Initially, 59 different LPS antigens were used (Table 1); later a further 7 LPS antigens, E.coli R1, R2, R3, R4, Re (F515) and O4 (Borstel strains) and E.coli Ra (ex O18), were added to fill the last (6th) plate of the set. The first 162 MAbs were tested in the 59 LPS ELISA, and the last 129 MAbs (fusions SZ-43, SZ-44, J5V, WN, VB, VN, X1SW) were tested in the full 66 LPS ELISA.

MAbs (hybridoma supernatants) were diluted 1:20 in phosphate-buffered saline pH 7.2 (Dulbecco A, Sigma) containing 0.01% (v/v) Tween 20, 0.05% sodium azide and 4% polyethylene glycol (PEG 2000, Sigma) and dispensed in 100m l aliquots in single rows across the 6 plate set. 8 MAbs were used in rows A to H respectively of each set of 6 plates, so that MAbs reacted only with a single well of each LPS-coated strip. Plates were incubated overnight (16h) at room temperature to allow maximal binding of weak antibodies, washed, and 100m l of a 1:200 dilution of urease-conjugated sheep anti-mouse Ig (IgG, IgM, IgA reactive) (SeraLab, England) was added to each well. Plates were incubated for 90min at 37°C, washed, rinsed in distilled water, and 100m l of urease substrate (SeraLab, England) was added. Colour was developed over 90 min at room temperature and changed from yellow (no reaction) to deep purple (strong positive), and was stopped by addition of thimerosal solution. Plates were read on a Multiskan MC (Labsystems, Finland) reader at 590nm, and results were collected by output from the reader to an IBM-compatible PC for further processing.

ELISA results were processed by a QuickBASIC (Microsoft) programme written in the laboratory which subtracted optical density (OD) readings of the negative control (BSA coated well) from each test (LPS coated well), and formatted results for transfer to a database (DBase IV, Borland) as net OD, where each record was comprised of a field for the antibody identifier and fields for each of the 66 LPS antigens. Results for each MAb were stored as sequential records, with blank fields to identify any LPS not assayed for a given MAb.

Correlation cluster segregation analysis and visualisation.

A program was written in QuickBASIC (Microsoft) to directly extract the stored data from the database, count and store the MAb identifiers (291) and LPS identifiers (66) as arrays, and assemble the OD results in a matrix (291x66). The correlation (Pearson's correlation coefficient, r) of pairs of MAbs over the set of 66 LPS antigens was determined for all MAb pairings and assembled in a 291x291 matrix. Similarly, the correlations of all pairings of LPS were determined over the 291 MAbs and assembled in a 66x66 matrix. Correlations excluded results where any pair combination included blank result fields, so that for certain MAbs correlations were calculated only on the 59 LPS antigens of the original ELISA set, and the 7 late-added LPS were only correlated over the later tested MAbs. Although their resolution is lessened by this, these results have been included for completeness.

Each set of MAb correlations was rearranged in its matrix as follows. Starting with the origin at the top, left position (row-1, column-1) (MAb-1 correlation with itself, r=1.0), the remaining MAb correlations to the right in row 1 were arranged in order of descending correlation, so that those most like MAb-1 were closest to it (high r) and those least like it were furthest from it (low r). The new order of MAbs in row 1 was noted, and the remaining whole rows (and the MAb identifiers) were rearranged so that column 1 reflected row 1 in order. The origin was then moved to row-2, column-2, and the remaining correlations to the right and below this position were rearranged as before, but correlations to the left and above were not rearranged. The process was repeated moving the origin stepwise down the diagonal from top left (1,1) to bottom right (291,291), rearranging correlations below and right of the origin but leaving arrangements of correlations above and left of the origin. This resulted into segregation along the diagonal of discrete clusters of "similar" MAbs.

The same process was applied to the matrix of LPS antigen correlations, from top left (1,1) to bottom right (66,66), resulting in clustering of serologically "similar" LPS.

Finally, the OD results matrix can be rearranged directly and/or a database can be constructed with the fields of each record arranged in order of the 66 rearranged LPS antigens and ELISA results re-read in according to the new order of rearranged MAbs, to complete the correlation-cluster segregation sort.

It is useful to construct maps of the rearranged correlations and ELISA (OD) results to visualise the relationships which have emerged. Both correlations and OD are continuous data, but can be digitised or banded into discrete subdivisions, and represented by colours (Figure 3) or shades of grey (Figures 4 and 5) for graphic representation. In this case, these were translated into a bitmaps which can be imported by various widely-used computer graphics software packages. In the figures shown the data was digitised and banded by a QuickBASIC programme written for this purpose, the bitmaps were pre-processed in Picture Publisher 3.1 (Micrografx), and the final figures were composed in Corel Draw 4 (Corel).

Results

MAb and LPS correlations.

As an example of MAb correlation relationships, the pairing of MAb SZ-043-27.07.03 with two other MAbs from the same fusion are shown in Figure 1 as a two-dimensional scatter of ELISA reactivities with different LPS, and the resultant correlations (r) and regression lines. The highest correlation of MAb SZ-043-27.07.03 is with MAb SZ-043-27.11.02 (r=0.85), and the ELISA reactivities are distributed around the regression line: these MAbs appear similar, and may be subclones of the same hybridoma since they arose from the same well (well 27) of the primary hybridoma screen. A high but lesser correlation (r=0.63) was obtained between MAb SZ-043-27.07.03 and MAb SZ-043-5.01.04: these antibodies are evidently different, both reacting strongly with a sub-group of the LPS, but MAb SZ-043-27.07.03 reacting more strongly with most other LPS than MAb SZ-043-5.01.04.

As an example of LPS correlation relationships, the pairing of LPS from E.coli O18 (K1-encapsulated, Bort) is shown with the LPS of E.coli R1 (k) and Serratia marcescens in Figure 2. The best correlation is between E.coli O18(K1+) LPS and the LPS of E.coli R1 (k) (r=0.81), where a group of MAbs react strongly with both LPS (possibly R1 core-type specific, indicating E.coli O18 expresses this core type). There is much less correlation between E.coli O18(K1+) LPS and the LPS of Serratia marcescens (r=0.41). Many more MAbs react strongly with E.coli O18(K1+) LPS than with the LPS of Serratia marcescens, while the other reactive MAbs vary in their degrees of reactivity with these LPS.

Correlation cluster segregation and ELISA rearrangement.

The maps of the rearranged MAb (C) and LPS (A) correlations and their relationship to the rearranged ELISA results (B) are shown.

The identities of the LPS are listed in rearranged order in Table 1, and the identities of the MAbs are listed in rearranged order in Table 2 (a and b). These may be related to the larger scale map of the ELISA results shown in Figure 4, which includes scales which relate to the sort numbers in these tables. There is clear congregation of LPS and MAbs into clusters along the identity diagonal which relate to ELISA reactivity patterns (Fig 3).

Relationships are also evident between families off the diagonal. These relationships may be positive (high correlation) where families are similar, or negative (high negative correlation) where families oppose in reactivity patterns, e.g. MAbs reacting predominantly with E.coli R2 oppose MAbs reacting predominantly with E.coli R1 when several examples of each LPS (rough and smooth) are present in the ELISA. The resolution of either LPS or MAbs into families is dependent on the variety of either in the ELISA: if a particular reactivity is not present the characteristics which determine it will not be resolved.

MAb WN1 222-5 is MAb 107

(Table: 1)

(Table: 2a)

(Table: 2b)

MAb family specificities for LPS core.

The major MAb families numerically and/or in ELISA reactivity pattern are listed in Table 2 (a and b) and their positions are marked on the sorted MAb correlation map (Figure 5). The principal characteristics of these families are as follows.

Family a:

Predominantly from fusion ES-184, these MAbs are E.coli O18 LPS serotype specific. All react with E.coli O18 K1+ (Bort) LPS, but some fail to react with its unencapsulated mutant: many also react with Serratia marcescens LPS. Some MAbs react with E.coli O18 (K1+) LPS, its rough mutant LPS (E.coli Ra exO18), and LPS from E.coli R1 (F470) and S.typhimurium Ra (strain ST1542) LPS. In general, these MAbs appear to have restricted specificity for this series of LPS.

Family b:

We believe these MAbs to be specific for the E.coli R1 core and cross-reactive on LPS expressing this core type: the reaction of some of them with S.typhimurium Ra (strain ST1542) LPS is unexplained. The smooth E.coli LPS with which they react are serotypes O16, O75, O2, K235, O4, O18 (K1+), and O128:B12.

Family c:

These antibodies appear to recognise Re chemotype LPS and show wide cross-reactivity with larger LPS, especially the "R1" type LPS seen by Family b MAbs. As well as E.coli and Salmonella LPS they bind to P.aeruginosa, Klebsiella, Vibrio cholera, Shigella and Serratia LPS. They bind weakly to monophosphoryl lipid A (ex S.minnesota Re) but not to diphosphoryl lipid A (ex E.coli Re D31m4)

Family d:

These are specific for the LPS from the rough mutant of E.coli O18(K1+).

Family e:

These MAbs recognise the Rc LPS chemotype, but not Rd2 or smaller LPS. Some, such as WN1 222.5, are widely cross-reactive with smooth E.coli, Salmonella and Shigella: others, such as SZ-43 27.11.2, are less cross-reactive and show strong reactivity predominantly with smooth LPS of E.coli "R1" type and some others.

Family f:

We have no interpretation of the binding pattern of these MAbs. They react with some Ra or Rb LPS from E.coli and Salmonella, and are not very cross-reactive. They are not strongly reactive in ELISA with any LPS.

Family g:

These MAbs are from a large family which shows a narrow range of binding activity for E.coli R2, R4 and C62 rough LPS and in some cases also for E.coli O127:B8 and O12 smooth LPS. Some also react with one strain of S.typhimurium Ra and smooth S.typhosa LPS.

Family h:

These are Re chemotype reactive MAbs which do not in general react with larger LPS, with the exception of E.coli C62 and S.typhimurium Ra strain ST1542 LPS.

Family i:

This group of MAbs appear to bind to E.coli R2 and R4 cores, with weak binding to R3 and K-12 cores. They also bind strongly to S.typhimurium Ra and moderately to S.minnesota Ra and S.typhimurium Rb. They bind to a range of smooth E.coli and all smooth Salmonella, which must share core serology.

Family j:

These MAbs are like Family i, but lack reactivity with E.coli O128, O127, O12, 55 and O26. Some MAbs react with Serratia marcescens and the unencapsulated E.coli O18(K1-) smooth LPS. While retaining Salmonella and E.coli R2 and R4 rough LPS reactivity, they predominantly react only with smooth LPS from Salmonella.

Family k:

These are very similar to Family b MAbs (E.coli R1 core type specific with smooth LPS cross-reactivity), but also have a weak reactivity with most LPS, including lipid A.

Family l:

These are restricted to E.coli R1 rough LPS and a small number of E.coli smooth LPS, especially E.coli O75.

Family m:

These very weak broadly cross-reactive MAbs may have some lipid A or Re LPS specificity.

Family n:

These show an unusual, restricted specificity for E.coli O127, O55, O26, O111 smooth LPS and S.minnesota O smooth LPS, with other minor weak binding activities.

Family o:

These are like Family n but show more extensive cross-reactivity, including reactivity with S.enteritidis and S.typhosa, and E.coli O15, O128, O127, O12 and O18(K-). They are not strongly reactive with rough LPS.

Family p:

These are rough Pseudomonas aeruginosa PAC605 specific.

Family q:

These MAbs are lipid A specific and broadly cross-reactive with all LPS (the missing reactivities in Figure 8 are LPS to which they were not tested in ELISA).

Family r:

These MAbs react with E.coli R2 and R3 rough LPS but may be E.coli R3 core type specific in smooth LPS, reacting with E.coli O86, O15, O26, O55, O111 and Shigella flexneri O(1A) smooth LPS.

Family s:

These overlap Family r specificity, but do not react with one of two E.coli O111 LPS. They also react with E.coli O128 LPS, and some react moderately with some Salmonella smooth LPS.

Family t:

These are Rc-reactive, broadly cross-reactive LPS like Family e, and may have segregated differently from Family e because they were not tested on the LPS added later to the ELISA series. They may also show some R3 core predominance, whereas Family e may show some R1 core predominance.

Family u:

These are weakly reactive MAbs with some Ra and S LPS reactivity for E.coli LPS which has no clear pattern.

Family v:

These are lipid A reactive MAbs with broad LPS cross-reactivity. They differ from Family q in showing better binding to rough LPS than to smooth LPS.

Family w:

These are Rc-specific LPS which do not bind well to larger LPS.

Family x:

These appear to bind smooth Salmonella LPS only.

Figure 6: MAb families

Figure 7: MAb families

Figure 8: MAb families

Figure 9: MAb families

Figure 10: MAb families

Other MAb reactivity patterns:

There are variations in binding patterns within MAb families. Figure 9 shows the progression in binding pattern across Family a (MAbs 1 to 15, Table 2) and includes MAb 291 (Table 2) which segregated away from Family a, apparently because of its additional reactivity with S.typhimurium Re (strain SL1181) LPS. These MAbs vary in reactivity with the two variants of E.coli O18 LPS from the K1 encapsulated and unencapsulated strains. Some react with Serratia LPS. The LPS structures which determine these reaction patterns are not known.

Figure 10 shows some individual MAb reaction patterns from MAbs not assigned to Families, and a progression across the Re-reactive Family h.

MAb ES-185 1.2.2 was raised against E.coli O6, but also reacts with S.typhimurium Ra (ST1542) and E.coli O16 LPS, and is contrasted with the anti-E.coli O18 MAbs from Family a (ES-184 series).

MAb X1 SW1 361.1 reacts with E.coli R4 core type only, and is not cross reactive.

MAb SZ-39 12.1 (MAb 290, Table 2) contrasts with the Re-reactive MAbs from Family h in showing Rd as well as Re reactivity. None react well with larger LPS, but cross-react with similar chemotype LPS from different species

LPS family seroclusters.

The LPS and lipid A used in this ELISA segregated into four main families on correlation cluster analysis which show little cross-correlation with each other (Figure 3).

The first main group can be divided into sub-groups (LPS Families a to c, Table 1) and is comprised of most of the incomplete core R-LPS and lipid A. The sorting process pushes three such LPS (Family g) to the end of the sorted series (Figure 3).

LPS Family a contains monophosphoryl lipid A and the deep rough Re LPS from S.minnesota, S.typhimurium (ST1102), Sh.flexneri and E.coli D31m4. It also contains Rd and Rb LPS from S.minnesota, Rb LPS from K.aerogenes, complete core Ra LPS from E.coli K-12, and smooth LPS from P.aeruginosa. These LPS evidently segregate together because the MAbs are unable to clearly differentiate between them serologically, and e.g. since none of these MAbs are particularly discriminatory for Pseudomonas LPS these have segregated to LPS clusters where they share serological features.

LPS Family b is comprised of Rc LPS from S.typhimurium and E.coli (J5), but excludes the Rc LPS of S.minnesota and P.aeruginosa PAC605 (Family g) which do not appear even to cross-correlate with Family b as seen by these MAbs (Figure 3).

LPS Family c is comprised of LPS from E.coli Rd2 (F583), Vib.cholera and S.typhimurium Re (SL1181), and cross-reacts with Family a LPS. The late-added E.coli Re (F515) LPS also segregates in Family c.

The second main LPS group has been designated Family d (Table 1) and contains E.coli R1 complete core Ra LPS (both strains), the E.coli Ra-LPS from the O18 rough mutant, and E.coli smooth LPS O16, O75, O2, K235, O4, O18(K1+), O6 and O128:B12. We interpret these as expressing the E.coli R1 core type.

The third main LPS group has been designated Family e (Table 1), and may consist of two overlapping groups. The first sub-group may include the E.coli R2 core serotypic LPS, and is comprised of smooth E.coli O127:B8 and O12, and also includes rough E.coli C62 and R4 (F2513) and rough S.typhimurium Ra (ST1542) which may overlap the second subgroup. These cross-correlate with two E.coli R2 LPS (both EH100) which probably belong to this family but have been sorted from it by the intervening late-added R-LPS from E.coli R3 (F673) and E.coli R4 (F2513) which have been tested with a more limited group of MAbs, and do not show any extensive cross-correlation (Figure 3). The second sub-group may include the Salmonella core serotypic LPS and is comprised of S.typhimurium Ra (TV119), S.typhimurium Rb (ST119), S.minnesota Ra (R60) and all the smooth Salmonella LPS.

The fourth main LPS group, Family f, is comprised of E.coli R3 (F673), smooth E.coli O86, O15, O55:B5, O111:B4 (two samples), O26:B6 and Sh.flexneri S-LPS, and may include the E.coli R3 core serotypic LPS.

The last group, Family g, comprising monophosphoryl E.coli lipid A (ex D31m4), S.minnesota Rc (R5) and P.aeruginosa Rc (PAC605) appears related to Family a by cross-correlation (Figure 3).

The LPS which do not fit into any of these major seroclusters are Serratia and E.coli O18(K1-) S-LPS, S.typhimurium Rd (ST1032) R-LPS and the late-added E.coli R2 (F576), R3 (F563) and R4 (F2513), which fall between the main correlation cluster groups (Figure 3).

Discussion

The correlation cluster segregation sorts the LPS into four predominant serological groups, two predominated by E.coli R1 or R3 core type serology respectively, one predominated by E.coli R2 and Salmonella core type serology and showing polarisation towards either, and one in which the inner core R-LPS congregate together with LPS whose only interaction with these 291 MAbs is in the inner core (Figure 3 and Table 1).

The MAbs also show congregation into predominant clusters along the identity diagonal (Figure 3), but there are more cross-correlations off the diagonal than with the LPS, indicating relationships between the MAb families. Numerically, the MAb families are dominated by groups which also appear related to LPS core-type serology (Figure 5). The largest group (Families r, s) appear to relate to the E.coli R3 core. Another large group appears related to the Salmonella core (Family j) and merges with the group related to the E.coli R2 core (Family i). The E.coli R1 core governs the clustering of Family b and the related Family k. Other predominant groups are Family a which are E.coli O18 serotype specific MAbs; and Family w which are Rc chemotype specific MAbs. A large group of MAbs (Families f to g) show a range of reactivities with certain unsubstituted cores (Ra) from E.coli and Salmonella, but little reactivity with complete S-LPS, and are difficult to assign to any known common serology. Some of the more interesting MAbs come from relatively small clusters. The largest of these clusters is Family t which contains the Rc-reactive MAbs which can also bind to larger LPS. Our assignation of LPS and MAb families has been guided by the correlation cluster sorting, but is subjective. The borders between families are not always distinct, there are often MAbs intermediate to these families which we have not assigned to any family (Figure 5), and within families there is variation. For the MAbs, this variation may reflect to some extent the potential of the polyclonal response to these LPS, with subtle shifts of fine specificity across major serological specificity groups. One of the interesting features of mapping the correlation matrices is that areas of negative cross-correlations can be found where MAb reactivities tend to show some degree of mutual exclusivity, i.e. one reacts with the antigens the other does not react with (Figure 3). The Family b, Family j and Family r MAbs show some negative cross-correlation with each other, which tends to confirm their specificity for mutually exclusive core serotypes. Some interesting associations arise from this. One such is the negative correlation between Family o MAbs and Family v MAbs. We now believe Family o MAbs recognise LPS-associated enterobacterial common antigen (ECA) (unpublished), while Family v MAbs are one of the lipid A reactive MAb families (Figures 7,8). This might infer that certain LPS expressing associated ECA have their ability to bind some anti-lipid A antibodies interfered with by the ECA.

Many of the 291 MAbs which were isolated fall into three major anti-LPS-core categories, (i) MAbs which react with inner core substructures only when these are exposed by core oligosaccharide chain breaks in mutant R-LPS or lipid A; (ii) MAbs which react with inner core substructures within more fully substituted LPS including intact complete core Ra-LPS and S-LPS; and (iii) MAbs which react with outer core structures, predominantly core serotypes. Other MAbs, such as the ES series of fusions, show O-specificity and were deliberately raised for this by immunisations with E.coli O18 (ES-184) or E.coli O6 (ES-185): the cross-reactivity of some anti-E.coli O18 MAbs with Serratia (Fig. 9) has been reported by others (24).

The MAbs which react with inner core substructures only when these are exposed are from MAb Families h (Re-specific) and w (Rc-specific) and MAb 290 (Table 2) (Rd-specific). Family p (Pseudomonas Rc-specific) shows inner core species restriction but falls into this category and contrasts with Pseudomonas LPS cross-reactive MAbs which we have isolated in another study (23). The weakly-reactive Family m MAbs may also belong to this category. Many such MAbs have been reported ( 19, 25-33), and may reflect the immunodominance of the lipid A-distal hydrophilic terminal residues of R-LPS immunogens in that such MAbs are frequently isolated. The frequency of their isolation may also reflect the tendency for the immunogen to be used as a primary screen antigen when selecting hybridomas for LPS reactivity by immunoassay. In our study we have always tested for cross-reactivity at primary screen, and have rejected many hybridomas showing immunogen selective specificity. This may explain the bias in the frequency of our retained MAbs which show cross-reactivity, and the relatively small numbers of such chemotype-specific MAbs.

MAbs which react with outer core structures, with little or no reactivity with R-LPS smaller than Rb chemotype, represent a large proportion of the 291 retained clones. These include a predominantly E.coli R1 core-type specific group (Families b and k, possibly Family l); a predominantly E.coli R3 core-type specific group (Families r and s) which also places Sh.flexneri in the R3 core type category as has been reported (34); and a more diffuse group ranging between E.coli R2 core specificity and Salmonella core specificity which probably reflects the similarities of the R2 and Salmonella core types (3). This group includes Families g, i, and j. MAbs from these families have been more extensively characterised in studies of a large panel of 60 of the 291 MAbs selected for specificity diversity, in ELISA against clinical isolate Gram-negative bacteria, which has been published in part (22, 21) and which will be presented in full by correlation cluster segregation analysis (Barclay et al, manuscript in preparation). In this analysis the core-type specificities and cross-reactivities dominate the segregation patterns.

Although it has been impossible to examine most of these MAbs in detail, because of their numbers, many of those which have been further characterised resemble the previous inner-core chemotype-specific group by exhibiting terminal residue specificity. These bind "naked" complete core (Ra) structures but fail to bind to fully substituted S-LPS as revealed by Western immunoblotting, where they bind only to the fast-migrating unsubstituted core: others show binding to the full S-LPS ladder as well unsubstituted core, and can evidently distinguish core-specific structures in the fully substituted S-LPS (22). The ELISA binding patterns (Figs 6-8) show that while some of these show exclusive reactivity with discrete groups of S-LPS and a particular R-LPS core type, others show similar discrete S-LPS group specificity but more cross-reactivity with naked R-LPS of different core types. Family i appears the most cross-reactive and encompasses Salmonella and E.coli "R2" LPS. Some of this family also react with R4, and K-12 R-LPS and some with certain LPS from the "R1" and "R3" groups. Further studies on clinical isolate bacteria have identified particular MAbs from this group which can distinguish apparent E.coli R1, R2, R3 and Salmonella core types in S-LPS. We have not identified MAbs with selective specificity for R4 or K-12 core types, except MAbs 243 to 245 (Table 2) which may be E.coli R4 core specific but which do not react with any of the other LPS in the ELISA.

None of these outer-core specific MAbs show full E.coli and Salmonella S-LPS cross reactivity, which may support the view that only the inner core structures share regions of similarity which may confer extensive cross-reactivity. Core-type specific MAbs with limited cross-reactivities have been reported from other studies (34-38) . Some of these show either the naked-core (Ra) specific or full O-chain-substituted core reactivity patterns in immunoblots (38) as found by us for different core-type specific MAbs (22) or cross-reactive MAbs (39). While most of these MAbs do not appear to show core-type cross-reactivity, MAb 786 of Peters et al (35) recognises an outer core structure common to E.coli K-12, B, R2 and R4, but not R1 or R3 core type LPS, which appears to have similar specificity to some MAbs from our MAb Families i and j.

MAbs which readily react with inner core substructures within more fully substituted LPS, including intact complete core Ra-LPS and S-LPS, represented a novel range of specificities when we first presented examples of these (21,36), and for us have been the most interesting in terms of their clinical potential. These MAbs are from three groups which appear to exhibit specificity for lipid A (Families q and v); Re (Family c); or Rc (Families e and t), and which also react with larger R-LPS and S-LPS. By this ELISA assay the lipid A and Re reactive MAbs show extensive cross-reactivity, while the Rc reactive MAbs tend to be more selective for E.coli, Salmonella and Shigella LPS and are not strongly reactive with the examples of Pseudomonas, Klebsiella, Serratia or Vib. cholera LPS in the assay.

There are many claims for wide cross-reactivity for anti-LPS-inner-core MAbs (40-43) and especially for the anti-lipid A MAbs E5 and HA-1A which have been used in clinical trials of sepsis (16,17). However, these MAbs seldom demonstrate cross-reactivity by conventional ELISA or blotting immunoassay techniques, and their ability to bind to free lipid A and also to R-LPS or S-LPS remains controversial (44,45). Recent studies tend to support the specific binding of E5 and HA-1A to lipid A (46-50). It is claimed that MAb HA-1A may also bind to R-LPS but not S-LPS in immunoblots, but shows binding to both R-LPS and at least some S-LPS by immune complex formation in liquid phase (47,50). MAb E5 may bind to lipid A and R-LPS by conventional ELISA and RIA, and may bind to a range of S-LPS from different species by immunolimulus assay (48,49). In either case, these anti-lipid A MAbs and those reported by Aydintug et al (43) appear different from those reported here in Families q and w whose anti-lipid A activity and R-LPS and S-LPS cross-reactivity is clearly demonstrable in our LPS-polymyxin complex ELISA (Figure 8). These reactivities have also been confirmed in the pure LPS ELISA described elsewhere (21) and on heat-killed bacteria (51). We have failed to detect specific binding of HA-1A in our assay systems (unpublished) which clearly differentiates HA-1A from our anti-lipid A MAbs, and the published reports of activity suggest that E5 also differs from our anti-lipid A MAbs.

The Re-specific LPS-cross-reactive MAbs from Family c were from a late fusion of the series (fusion SZ-43) following cyclical immunisation with heat-killed E.coli complete core (Ra) bacteria of different core types, including the rough mutant of E.coli O18, and have not been extensively characterised. In this ELISA they react strongly with S-LPS of the E.coli R1 core-type group, but show good reactivity with most LPS. They react well with Re LPS from S.minnesota, S.typhimurium and E.coli F515, but less well with Re LPS from E.coli D31m4. They have some weak reactivity with monophosphoryl lipid A (ex S.minnesota Re) but no reactivity with diphosphoryl lipid A (ex E.coli D31m4). It may be that their binding site overlaps the KDO and lipid A regions: however, this binding site is apparently exposed on most LPS. Many anti-Re MAbs have been reported by Appelmelk et al which were raised following immunisation with Re-LPS from S.minnesota 595 (27,52) and which are predominantly chemotype-specific. Some show reactivity with larger LPS and may react with the terminal KDO of the KDO trisaccharide (27), and are therefore probably different from those reported here.

The Rc-specific LPS-cross-reactive MAbs from Families e and t represent novel anti-LPS MAbs in that their minimal binding structure is Rc and they bind to larger LPS containing this structure. There are a range of MAbs in these families and not all have been characterised. There is some variation in cross-reactivity, with some MAbs from Family e showing some preference for the E.coli R1 core type. Some of these MAbs have been well characterised (21,22,39,51,53) and have been the subject of much unpublished work. In general, where these MAbs are able to bind to R-LPS or S-LPS they will neutralise the endotoxic activity of that LPS in vitro, e.g. by inhibition of LAL activity, blocking of LPS-stimulated IL-6 or TNF responses of mouse and human leukocytes; or in vivo, e.g. inhibition of LPS-induced fever in rabbits, blocking of the lethal effects of LPS in the galactosamine-treated mouse model, and are effective in a variety of other models of endotoxin activity. In general they are not bactericidal and are not effective in conventional models of bacteraemia. However, their binding to antibiotic treated bacteria (53) suggest their potential should be explored in animal models of bacteraemia with different antibiotics. There is some similarity between this group of MAbs to the MAb MM3 described by Nnalue et al (54) which binds to the heptose region of the LPS core and shows considerable cross-reactivity with larger LPS from Salmonella, but is restricted in its E.coli S-LPS cross reactivity.

There are a variety of MAbs which occur with low frequency and do not fall into any family group. There are also families whose reactivity profile is not readily interpretable currently. Our focus was to isolate MAbs which showed both extensive cross reactivity for LPS and a capacity to block endotoxin biological activity. The Rc-specific LPS-cross-reactive MAbs go a long way to meeting these specifications. The endotoxin inhibiting effects of others of the LPS-core substructure-specific S-LPS cross-reactive MAbs has not been evaluated. Many of these MAbs would appear to have interesting potential for clinical development, some have excellent performance on preclinical evaluation, and all appear to differ from any MAb which has been tested clinically so far. Despite the controversy regarding the clinical efficacy of the anti-lipid A MAbs E5 and HA-1A in Gram-negative sepsis (18,44) we believe that the concept of anti-endotoxin immunotherapy may still be valid. None of the cytokine-modulating or other novel therapies appears to have met with any more success than the anti-lipid A immunotherapies. It may be appropriate to try anti-endotoxin antibody therapy again with more judiciously selected MAbs such as some of those described here.

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Legends:

Table 1

: The LPS used in ELISA in correlation-cluster sorted order. Strain and source are indicated (see methods). The assigned family groups are given (fam).

Table 2

: The 291 anti-LPS monoclonal antibodies tested in ELISA in correlation-cluster sorted order. The assigned family groups are given.

Figure 1

: Scattergram of MAb SZ-043-027.07.03 ELISA OD versus MAbs SZ-043-027.11.02 and SZ-043-005.01.04 ELISA ODs over the 66 LPS antigens. Correlation (Pearson's r) and regression lines are shown.

Figure 2

: Scattergram of E.coli O18(K1+) LPS ELISA OD versus E.coli R1(k) LPS and Serratia marcescens LPS ELISA ODs over the 291 anti-LPS MAbs. Correlation (Pearson's r) and regression lines are shown.

Figure 3

: Map of the rearranged LPS antigen correlations (A) and anti-LPS MAb correlations (C), and the resultant rearranged ELISA OD (B) following correlation-cluster sorting of the original ELISA results. The LPS and MAb orders are as indicated in Tables 1 and 2.

Figure 4

: The rearranged ELISA OD results with LPS antigen sort numbers (horizontal) and MAb sort numbers (vertical) as indicated in Tables 1 and 2. The OD scale (intensity) is as for Figure 5.

Figure 5

: Map of rearranged correlations and ELISA results (see legend for Fig. 3). Assigned MAb family groups are indicated (a to x) as in Table 2.

Figure 6

: ELISA reactivity patterns of two MAbs from each of familiesa to h. The LPS antigens are as indicated in Table 1. The ELISA OD scale is 0.0 to 2.0.

Figure 7:

ELISA reactivity patterns of two MAbs from each of familiesi to p. See legend for Fig 6.

Figure 8:

ELISA reactivity patterns of two MAbs from each of familiesq to x. See legend for Fig 6.

Figure 9:

ELISA reactivity patterns of pairs of MAbs from family a (anti-E.coli O18) showing a progressive change in reactivity across this family. See legend for Fig 6.

Figure 10

: Some unassigned MAb reactivity patterns (see legend for Fig 6). MAb ES-185 1.2.2 (anti-E.coli O6 LPS) compared to MAbs from the ES-184 series (Family a, anti-E.coli O18 LPS); the reaction pattern of MAb X1 SW1 361.01 (E.coli R4 (b) reactive); MAb SZ-39 12.1 (Rd and Re reactive) and Family h MAbs (Re reactive).

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