G. Robin Barclay PhD

E-mail: r.barclay@virgin.net

We have reported extensively on depressed EndoCAb levels in sepsis and endotoxaemia, and a number of other studies using different assays have come to similar conclusions, e.g. Nys et al (3). As Strutz et al have confirmed for their patients (1), the deficiencies of EndoCAb are associated with risk of poor outcome. This relationship is not strict, and poor outcome could not be predicted solely on the basis of reduced EndoCAb. Nevertheless, it may be that the EndoCAb assay might predict which patients could benefit from passive immunotherapy since there would seem to be no point in administering passive antibody to patients already displaying substantial endogenous levels of that antibody.

Clinical trials in sepsis are always likely to be unwieldy because before a diagnosis can be made the patient will have experienced prodromal sepsis which may significantly perturb any baseline measurements, initiate the cytokine cascade, and induce changes in immunity. Sequential studies can follow these changes, while single sample studies may be more difficult to interpret in this perturbed environment. We do not understand what determines an individual’s stable levels of IgG or IgM EndoCAb. Recent studies by us have indicated that in patients awaiting cardiopulmonary bypass surgery the preoperative IgM EndoCAb in patients with a poor outcome following surgery is significantly lower than preoperative levels in patients with an uneventful outcome (5, 6), and differences in IgG EndoCAb reach significance during the procedure when perioperative falls in EndoCAb are seen (5). Cardiopulmonary bypass patients may present better subjects for study than patients already perturbed by sepsis for future trials of anti-endotoxin therapies. The EndoCAb assay could reveal which patients might benefit from such therapies to increase the power and decrease the cost of any future study.

The association of low EndoCAb with poor outcome in sepsis begs the question: which therapeutic agent should be used to attempt to intervene in these various clinical presentations of endotoxin pathogenesis? The answer is - there are currently no intervention treatments indicated and available for general use other than the standard treatments. The immunotherapies based on cross reactive antibodies to the core region of endotoxin are largely discredited (7), none of the agents designed to interrupt the post-endotoxin cytokine cascade have stood up to clinical trial in sepsis, and the new non-antibody endotoxin-neutralising agents such as bactericidal/permeabilty-increasing protein (BPI) which already hold out some promise of efficacy (8) are still at the stage of clinical evaluation. Other inhibiting strategies, such as blocking the availability to endotoxin of the leucocyte CD14 receptor (9) are still only at the experimental stage. Around 30 years of research has gone into the search for immunotherapy based on cross-reactive antibodies to the core region of endotoxin. Unless the reported antibody depressions are simply a coincidental phenomenon, it is worth speculating on why no anti-endotoxin-core immunotherapeutic antibody agent has been found. It may be that many adequate antibodies have been overlooked or inappropriately tested, but is also true that many wholly inappropriate antibodies have been inadequately characterised and over-strenuously promoted to the detriment of comprehension in this somewhat anarchic field of study.

The antibody preparations which have been available for clinical evaluation can be categorised as (i) polyvalent normal immunoglobulins; (ii) polyvalent hyperimmune immunoglobulins, subcategorised into (a) hyperimmune "natural" antibodies found by identifying high-titre donors with pre-existing immunity, and (b) hyperimmune "elicited" antibodies acquired following immunisation of donors with various LPS immunogens; and (iii) monoclonal antibodies (MAb). The immunoglobulins have been delivered as plasma, gammaglobulin (IgG) or immunoglobulins with IgG, IgA and IgM content (IgGAM), so that there is a wide variety of antibody presentation modes to attempt to reconcile.

A number of studies have demonstrated that normal pooled gammaglobulin or immunoglobulins (IgGAM) can improve outcome in sepsis-related conditions (10, 11, 12). Although it has been inferred that immunoglobulin preparations containing IgM are superior to gammaglobulin preparations because the IgM component contains bactericidal antibodies to O-specific side chain of the LPS (13), direct comparison of such preparations do not suggest superiority of either preparation (12). However, there are a number of studies of normal immunoglobulins in which no benefit has been found, and the predominant view is that the protective effects of normal polyvalent immunoglobulins are at best marginal or confined to some subgroup of patients whose characteristics remain unrecognised. Without consensus, polyvalent normal immunoglobulin is not generally indicated for therapy in these clinical conditions.

The E.coli J5-immune plasma and gammaglobulin reported by Ziegler at al. are the only clinical studies of elicited hyperimmune antibody to endotoxin core, where some benefit was found using immune plasma but little benefit was found when immune gammaglobulin was used (14). Attempts to rationalise this have concluded that relevant cross-protective antibodies may not have been elicited by the J5 R-LPS immunisation (15). There is more convincing evidence of protective activity in clinical studies of gammaglobulins hyperimmune in "natural" anti-endotoxin antibodies by Gaffin et al (16) and Fomsgaard et al (17). Although these employed cocktails of multiple smooth enterobacterial LPS to identify donor plasma with high endotoxin antibody content, our experience predicts that they probably selected plasma enriched in EndoCAb, resulting in hyperimmune gammaglobulin very similar to our EndoCAb gammaglobulin which was not evaluated for efficacy in clinical studies, but showed significant protection in a sheep model of E.coli septicaemia (4). Nevertheless, despite encouraging results, none of these naturally-hyperimmune gammaglobulins remain in production for clinical use.

One of the main reasons that polyclonal immunoglobulin therapy was not widely pursued is that the monoclonal endotoxin-core-specific antibodies came to dominate this research in the 1980’s. When MAbs failed to deliver effective therapy, general disillusion set in with the concept of the therapeutic potential of endotoxin-core cross-reactive antibodies. However, despite popular perception, no relevant MAb with endotoxin-core cross-reactive properties has ever been evaluated clinically. It is probable that many attempts to raise these MAbs used inappropriate antigens, as may have been the case for the preceding studies of immune polyclonal sera (15). The popular small R-LPS immunogens, while sharing primary structures across species, differ from commonly encountered pathogenic endotoxin in secondary and tertiary structure, and contain novel epitopes where the components of complete LPS would normally be attached. This resulted in many immunogen-specific MAbs with poor cross reactivity for other LPS (18). The much publicised lipid A-specific MAbs E5 and HA-1A, which were the only MAbs subjected to clinical trial for sepsis immunotherapy, neither bind nor neutralise any endotoxin (19), and it can be argued that more rigorous preclinical evaluation should have aborted their development before they reached the stage of a clinical evaluation where they predictably failed. It is ironic that these anti-lipid A MAbs which have determined the current perception of the efficacy of anti-endotoxin-core antibodies cannot themselves be described as anti-endotoxin-core antibodies. However, this description persists and reinforces the bias.