Novel IgG-degrading enzymes of the IgdE protease family link substrate specificity to host tropism of <i>Streptococcus</i> species

Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact

Complement activation by immunoglobulin does not depend solely on C1q binding.

A matched set of rat chimeric antibodies has been studied for their ability to activate various key stages of the complement cascade. Rat IgM and IgG2b were efficient at all stages from C1q binding to cell lysis. However, for other isotypes, a direct correlation between C1q binding and cell lysis did not apply. IgG2a, which was only modestly efficient at C1q binding, was relatively more so for binding and activation of while C1, and was by far the most effective isotype after IgG2b and IgM for C4 and C3 binding. IgG2c was relatively efficient at binding C1q and C1, but less so for the binding of C4 or for later stages. IgA was efficient at binding C1, but again, this was not reflected in activation of later stages. The results suggest that properties of different isotypes, as well as influencing binding of C1q, may regulate attachment of the C1r2C1s2 tetramer. In addition, distinct features of certain isotypes may favor C4 activation and binding, independent of their ability to activate C1

Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies.

Lysis of human lymphocytes by autologous complement had been studied using a range of monoclonal antibodies against different antigens. Antigen specificity (and not antibody isotype) was the most important factor which influenced cell lysis and this could not be accounted for merely by differences in surface density between antigens. Three antigens with comparable surface density were studied in detail: CAMPATH-1 (lytic), major histocompatibility complex class I (lytic) and leukocyte common antigen (poorly lytic). C1q binding was roughly proportional to antibody binding and dependent on antibody isotype. However, the lytic antibodies were much better able to bind and activate whole C1 than the poorly lytic ones. This result would not have been predicted from traditional concepts of complement activation but can be interpreted in the light of models for C1 activation which involve Fc-Fc interactions, Fc-C1r2s2 interactions and a critical C1q stem-arm angle for C1 binding and activation

Therapeutic potential of monoclonal antibodies to the leukocyte-common antigen. Synergy and interference in complement-mediated lysis.

A series of rat monoclonal antibodies against the human leukocyte-common antigen were isolated and, by means of competitive binding assays with purified antigen, two distinct groups were defined that recognize different epitopes of the molecule. None of these antibodies were lytic with human complement, but when antibodies against each of these two epitopes were used in combination, synergistic lysis with human complement could be obtained. Synergistic lysis was only seen when each antibody of the pair was of the IgG2b subclass. IgG2a antibodies could not synergize, and in fact could interfere with lysis obtained by the pair of IgG2b antibodies. Although synergy has so far only been studied for rat monoclonal antibodies we also show that it is possible with mouse antibodies or with combinations of mouse and rat antibodies. The information about the principles of synergy and its interference should provide a rationale for using well-planned cocktails of monoclonal antibodies for therapy rather than a shotgun polyclonal antiserum

Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q.

Humanized antibodies are likely to have a major role in therapy and it is important to define their interaction with physiological effectors. By comparing a matched series of chimeric human mAbs we found that igG1 was most efficient in complement lysis, although IgG3 bound more C1q. To resolve this paradox we compared the ability of human IgG1, IgG2, IgG3, IgG4, and IgE and rat IgG2b to cause C1q binding, C1 binding and activation, C4 activation, C4b binding, and C3b binding. Rat IgG2b was included because this isotype has already successfully been used for therapy. Human IgG1 was less efficient than IgG3 and fixing C1q and C1 on the cell surface, but the number of C4 molecules bound per C1 was 10-fold greater for IgG1 than for IgG3. This difference, amplified through later stages of the complement cascade, can account for the superiority of IgG1 for cell lysis. The efficiency of IgG1 in fixing C4 was not due to a favored binding site on the antibody molecule, since virtually all of the bound C4b was attached to the cells. Rather, it appeared that the activation of C4 by C1s was greatly favored by IgG1 compared with IgG3. It should be possible to combine the optimal properties of IgG1 and IgG3 antibodies to produce an improved therapeutic reagent

Synergistic complement lysis by monoclonal antibodies to the human leukocyte common antigen requires both the classical and alternative pathways.

In a previous study we isolated a series of rat monoclonal antibodies to the human leukocyte common (LC) antigen and demonstrated that synergistic complement lysis was possible between IgG2b antibodies which recognised different epitopes. In this report we have examined the mechanisms that were involved in synergistic lysis. We found that the number of C1q binding sites increased from 30,000 to 40,000/cell using a single antibody to 90,000/cell when two IgG2b antibodies to different epitopes were used together. The affinity of C1q binding also increased approx. 3-fold for the synergistic pair, and there was a similar increase in the rate of C1 activation. Combinations of an IgG2b with IgG1 or IgG2a gave much smaller increases in the amount of C1q bound and in the rate of C1 activation. Despite the large number of C1q molecules bound with the optimal synergistic pair and the increased rate of C1 activation, lysis was inefficient in serum depleted of Factor D, suggesting a requirement for the alternative pathway. This is the first demonstration of the need for the alternative pathway in complement lysis by monoclonal antibodies

Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.

Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications

Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance.

Allogeneic reactions are the major limitation to organ transplantation. These are manifested as rejection of the grafted tissue, and also, in the case of bone marrow transplantation (BMT), graft-versus-host disease (GVHD). Recent methods of avoiding GVHD, by depleting T cells from donor marrow, have led to an increased incidence of marrow graft rejection. Current recipient conditioning protocols involving drugs or irradiation cannot safely be increased, so alternatives must be found. Monoclonal antibodies can be used to control immune responses in vivo, and would be useful in this context if we could define and deplete the cells responsible for marrow rejection. We show here that elimination of residual L3T4+ and Lyt-2+ cells from mice receiving fully mismatched bone marrow abrogates rejection and promotes tolerance to donor-type skin grafts, even in sub-lethally irradiated recipients