David J. Seilly

Streptococcal Inhibitor of Complement Inhibits Two Additional Components of the Mucosal Innate Immune System: Secretory Leukocyte Proteinase Inhibitor and Lysozyme

ABSTRACT Streptococcal inhibitor of complement (SIC) is a 31-kDa extracellular protein of a few, very virulent, strains of Streptococcus pyogenes (particularly M1 strains). It is secreted in large quantities (about 5 mg/liter) and inhibits complement lysis by blocking the membrane insertion site on C5b67. We describe investigations into the interaction of SIC with three further major components of the innate immune system found in airway surface liquid, namely, secretory leukocyte proteinase inhibitor (SLPI), lysozyme, and lactoferrin. Enzyme-linked immunosorbent assays showed that SIC binds to SLPI and to both human and hen egg lysozyme (HEL) but not to lactoferrin. Studies using 125I-labeled proteins showed that SIC binds approximately two molecules of SLPI and four molecules of lysozyme. SLPI binding shows little temperature dependence and a small positive enthalpy, suggesting that the binding is largely hydrophobic. By contrast, lysozyme binding shows strong temperature dependence and a substantial negative enthalpy, suggesting that the binding is largely ionic. Lysozyme is precipitated from solution by SIC. Further studies examined the ability of SIC to block the biological activities of SLPI and lysozyme. An M1 strain of group A streptococci was killed by SLPI, and the antibacterial activity of this protein was inhibited by SIC. SIC did not inhibit the antiproteinase activity of SLPI, implying that there is specific inhibition of the antibacterial domain. The antibacterial and enzymatic activities of lysozyme were also inhibited by SIC. SIC is the first biological inhibitor of the antibacterial action of SLPI to be described and may prove to be an important tool for investigating this activity in vivo. Inhibition of the antibacterial actions of SLPI and lysozyme would be advantageous to S. pyogenes in establishing colonization on mucosal surfaces, and we propose that this is the principal function of SIC.

Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes.

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme‐linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b‐9, but only weakly to individual complement components. The implications of these data for virulence of SIC‐positive streptococci are discussed, in light of the fact that Gram‐positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC.

The interaction of streptococcal inhibitor of complement (SIC) and its proteolytic fragments with the human beta defensins.

Streptococcal inhibitor of complement (SIC) is a 31 kDa extracellular protein produced by a few highly virulent strains of Streptococcus pyogenes (in particular the M1 strain). It has been shown additionally to inhibit four further components of the mucosal innate response—lysozyme, secretory leucocyte proteinase inhibitor, human α‐defensin 1 and the cathelicidin LL‐37 which are all bactericidal against Group A Streptococci (GAS). We now show that SIC also inhibits variably the antibacterial action of hBD‐1, ‐2 and ‐3. By enzyme‐linked immunosorbent assay (ELISA), SIC binds strongly to hBD‐2 and hBD‐3, but not at all to hBD‐1. Investigation of the antimicrobial action of β‐defensins hBD‐1, ‐2 and ‐3 against GAS in two different buffer systems shows that both the killing efficiencies of all three defensins, and the binding of SIC to them, occurs more efficiently in 10 mm Tris buffer than in 10 mm phosphate. The lower ionic strength of the Tris buffer may underlie this effect. hBD‐1 kills the M1 strain of GAS only in 10 mm Tris, but is able to kill an M6 (SIC negative) strain in 10 mm phosphate. The inhibition of hBD‐3 by SIC is clearly of physiological relevance, that of hBD‐2 is likely to be so, but the inhibition of hBD‐1 occurs only at lower ionic strength than is likely to be encountered in vivo. Elastase digestion of SIC yields three major fragments of MW 3·843 kDa comprising residues 1–33 (fragment A); 10·369 kDa comprising residues 34–126 (fragment B); and MW 16·487 kDa, comprising residues 127–273 (fragment C). By ELISA, only fragment B binds to hBD‐2 and hBD‐3 and this may indicate the inhibitory portion of the SIC molecule.

Subversion of the innate immune response by micro-organisms.

A microbe becomes a pathogen by successfully evading the host’s immune responses, and the microbial strategies for so doing are legion. They include methods to avoid recognition by the immune system—for example, by antigenic variation as shown by influenza and HIV and by parasites or plasmodia, or by acquiring a host coat as is done by worms and retroviruses. Another major mechanism is to avoid the effector mechanisms of the immune response. This can be done by subverting cytotoxic T cells by the production of decoy HLA molecules; or by subverting Fc function by producing Fc receptor homologues; or by subverting complement by producing homologues of complement control proteins (CCPs). Some viruses also have developed methods of subverting apoptosis in the cells that they infect. This paper concentrates on the innate immune response. The definition of this term is a little fuzzy. Fearon and Locksley regard all mechanisms using germline coded molecules as being innate which therefore includes natural antibodies with germline V regions.1 Perhaps a more conventional definition is that innate mechanisms are those that are not specifically altered by prior exposure to the same pathogen. In the first part of this paper some examples of subversion of the complement system will be described. The second half describes some much newer work from our laboratory that takes us into aspects of the innate immune response on mucosal surfaces that have not so far been described. Figure 1 shows a greatly simplified view of complement activation by micro-organisms. Regulation occurs principally at two points—the first is action of the C3 converting enzymes and the second, action of the membrane attack complex (MAC). Figure 1 Principal activities of the complement system. Regulation of the C3 converting enzymes is produced by a number of proteins (fig 2), all of which are based on …

Mixed Adjuvant Formulations Reveal a New Combination That Elicit Antibody Response Comparable to Freund's Adjuvants

Adjuvant formulations capable of inducing high titer and high affinity antibody responses would provide a major advance in the development of vaccines to viral infections such as HIV-1. Although oil-in-water emulsions, such as Freunds adjuvant (FCA/FIA), are known to be potent, their toxicity and reactogenicity make them unacceptable for human use. Here, we explored different adjuvants and compared their ability to elicit antibody responses to FCA/FIA. Recombinant soluble trimeric HIV-1 gp140 antigen was formulated in different adjuvants, including FCA/FIA, Carbopol-971P, Carbopol-974P and the licensed adjuvant MF59, or combinations of MF59 and Carbopol. The antigen-adjuvant formulation was administered in a prime-boost regimen into rabbits, and elicitation of antigen binding and neutralizing antibodies (nAbs) was evaluated. When used individually, only FCA/FIA elicited significantly higher titer of nAbs than the control group (gp140 in PBS (p<0.05)). Sequential prime-boost immunizations with different adjuvants did not offer improvements over the use of FCA/FIA or MF59. Remarkably however, the concurrent use of the combination of Carbopol-971P and MF59 induced potent adjuvant activity with significantly higher titer nAbs than FCA/FIA (p<0.05). This combination was not associated with any obvious local or systemic adverse effects. Antibody competition indicated that the majority of the neutralizing activities were directed to the CD4 binding site (CD4bs). Increased antibody titers to the gp41 membrane proximal external region (MPER) and gp120 V3 were detected when the more potent adjuvants were used. These data reveal that the combination of Carbopol-971P and MF59 is unusually potent for eliciting nAbs to a variety of HIV-1 nAb epitopes.

A Fusion Intermediate gp41 Immunogen Elicits Neutralizing Antibodies to HIV-1

Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp41int folds into a compact elongated structure that induces neutralizing antibodies upon immunization. Conclusion: Presentation of gp41int in a lipid environment is beneficial to induce neutralizing antibodies. Significance: Membrane-anchored gp41int is a promising antigen to improve breadth and potency of anti-gp41 antibody responses. The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41int-Cys) and show that it folds into an elongated ∼12-nm-long extended structure based on small angle x-ray scattering data. Gp41int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140CA018 was higher than that induced by gp41int-Cys, the majority of animals immunized with gp41int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.

Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system

S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

Complotype affects the extent of down‐regulation by Factor I of the C3b feedback cycle in vitro

Sera from a large panel of normal subjects were typed for three common polymorphisms, one in C3 (R102G) and two in Factor H (V62I and Y402H), that influence predisposition to age‐related macular degeneration and to some forms of kidney disease. Three groups of sera were tested; those that were homozygous for the three risk alleles; those that were heterozygous for all three; and those homozygous for the low‐risk alleles. These groups vary in their response to the addition of exogenous Factor I when the alternative complement pathway is activated by zymosan. Both the reduction in the maximum amount of iC3b formed and the rate at which the iC3b is converted to C3dg are affected. For both reactions the at‐risk complotype requires higher doses of Factor I to produce similar down‐regulation. Because iC3b reacting with the complement receptor CR3 is a major mechanism by which complement activation gives rise to inflammation, the breakdown of iC3b to C3dg can be seen to have major significance for reducing complement‐induced inflammation. These findings demonstrate for the first time that sera from subjects with different complement alleles behave as predicted in an in‐vitro assay of the down‐regulation of the alternative complement pathway by increasing the concentration of Factor I. These results support the hypothesis that exogenous Factor I may be a valuable therapeutic aid for down‐regulating hyperactivity of the C3b feedback cycle, thereby providing a treatment for age‐related macular degeneration and other inflammatory diseases of later life.

Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrPC). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrPSc followed by detection with a europium-labelled anti-PrPC antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrPSc in cases of vCJD.

Further studies of the down-regulation by Factor I of the C3b feedback cycle using endotoxin as a soluble activator and red cells as a source of CR1 on sera of different complotype.

In this paper we have extended our earlier studies of the action of increasing Factor I concentration on complement activation by using a soluble activator, lipopolysaccharide (LPS) endotoxin, and using human erythrocytes as a source of CR1 – the co‐factor needed for the final clip of iC3b to C3dg by Factor I. Using this more physiological system, the results show that we can predict that a quite modest increase in Factor I concentration – 22 µg/ml of extra Factor I – will convert the activity of the highest risk sera to those of the lowest risk. Preliminary experiments have been performed with erythrocytes allotyped for CR1 number. While we have not been able to perform an adequate study of their co‐factor activities in our assays, preliminary experiments suggest that when Factor I levels are increased the difference produced by different allotypes of red cells is largely overcome. This suggests that in patients with paroxysmal nocturnal haemoglobinuria (PNH) treated with eculizumab, additional treatment with Factor I may be very useful in reducing the need for blood transfusion. We have also explored the age‐related allele frequency for the two polymorphisms of Factor H and the polymorphism of C3. In our population, unlike the 1975 study, we found no age variation in the allele frequency in these polymorphisms. This may, however, reflect that the Cambridge BioResource volunteers do not include many very young or very elderly patients, and in general comprise a population not greatly at risk of death from infectious disease.