Inhibit Complement Deposition Research Articles

Immune interface interference vaccines: An evolution-informed approach to anti-bacterial vaccine design.

Developing protein-based vaccines against bacteria has proved much more challenging than producing similar immunisations against viruses. Currently, anti-bacterial vaccines are designed using methods based on reverse vaccinology. These identify broadly conserved, immunogenic proteins using a combination of genomic and high-throughput laboratory data. While this approach has successfully generated multiple rationally designed formulations that show promising immunogenicity in animal models, few have been licensed. The difficulty of inducing protective immunity in humans with such vaccines mirrors the ability of many bacteria to recolonise individuals despite recognition by natural polyvalent antibody repertoires. As bacteria express too many antigens to evade all adaptive immune responses through mutation, they must instead inhibit the efficacy of such host defences through expressing surface structures that interface with the immune system. Therefore, 'immune interface interference' (I3) vaccines that target these features should synergistically directly target bacteria and prevent them from inhibiting responses to other surface antigens. This approach may help us understand the efficacy of the two recently introduced immunisations against serotype B meningococci, which both target the Factor H-binding protein (fHbp) that inhibits complement deposition on the bacterial surface. Therefore, I3 vaccine designs may help overcome the current challenges of developing protein-based vaccines to prevent bacterial infections.

Engineering human pluripotent stem cell lines to evade xenogeneic transplantation barriers

Successful allogeneic human pluripotent stem cell (hPSC)-derived therapies must overcome immunological rejection by the recipient. To build reagents to define these barriers, we genetically ablated β2M, TAP1, CIITA, CD74, MICA, and MICB to limit expression of HLA-I, HLA-II, and natural killer (NK) cell activating ligands in hPSCs. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit NK cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of NK cells also led to persistent teratomas. Thus, Tcell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to define cell type-specific immune barriers and conduct preclinical testing in immunocompetent mouse models.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Corrigendum

Cellular MicrobiologyVolume 23, Issue 1 e13286 CORRIGENDUMFree Access Corrigendum This article corrects the following: Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival Jennifer A. Caine, Yi-Pin Lin, Julie R. Kessler, Hiromi Sato, John M. Leong, Jenifer Coburn, Volume 19Issue 12Cellular Microbiology First Published online: September 26, 2017 First published: 09 November 2020 https://doi.org/10.1111/cmi.13286AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In “Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival”, by Caine et al. (2017)which was published in volume 19, issue 12, September 2017, the authors wish to notify readers that we have not been able to reliably reproduce the data in Figure 5. The corresponding text “To determine if OspC is able to directly inhibit the complement cascade allowing WT B. burgdorferi to survive in the bloodstream of mice, we performed an ELISA-based complement activation assay. Plates were coated with initiators of each pathway of the complement cascade; Immunoglobulin M (IgM; classical pathway), mannan (lectin pathway), lipopolysaccharide (LPS; alternative pathway), or buffer (negative control). Coated plates were incubated with a mixture of pooled active human serum and various concentrations of purified GST-OspC B31, GST, or buffer, and complement deposition was measured in each well by ELISA (Figure 5). Wells coated with IgM or mannan and incubated with 1000 nM GST- OspC have significantly lower levels of deposited complement compared to wells incubated in GST (Figure 5a,b). This effect is not seen in LPS coated wells, where the addition of GST-OspC does not inhibit complement deposition compared to GST at any concentration tested (Figure 5c) and Figure 5” should be removed from the paper. In addition, Experimental Procedures section 4.13 “Complement Inhibition ELISAs” should be removed. All other data shown are reproducible, so our primary conclusion is supported. REFERENCE Caine, JA, Lin, Y-P, Kessler, JR, Sato, H, Leong, JM, and Coburn, J. Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival. Cellular Microbiology, 2017; 19:e12786. PMID 28873507, PMCID PMC5680108. https://doi.org/10.1111/cmi.12786 Volume23, Issue1January 2021e13286 ReferencesRelatedInformation

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Polyclonal IgM and IgA block in vitro complement deposition mediated by anti-ganglioside antibodies in autoimmune neuropathies

Intravenous immunoglobulin (IVIG), consisting of IgG, is the first-line treatment for Guillain–Barré syndrome and multifocal motor neuropathy. IgG, but neither IgM nor IgA, has been demonstrated in vitro to inhibit complement deposition mediated by anti-ganglioside autoantibodies in sera from patients with both conditions. The objective of this study is to investigate the in vitro effectiveness of IgM and IgA in inhibiting complement deposition to ganglioside/anti-ganglioside antibody complexes. Serum samples were obtained from patients with multifocal motor neuropathy associated with anti-GM1 IgM antibodies, Guillain–Barré syndrome associated with anti-GM1 IgG antibodies and Miller Fisher syndrome associated with anti-GQ1b IgG antibodies. Inhibition of complement deposition using different immunoglobulin preparations was measured by enzyme-linked immunosorbent assay. IgM/A-enriched IVIG and immunoglobulin isotypes (polyclonal IgM and IgA) showed higher potential in inhibiting complement deposition than standard IVIG. Although the safety concerns about the use of IgM and IgA for an immunotherapy still remain, IgM and IgA may serve as an alternative immunotherapy in those anti-ganglioside antibody-mediated neuropathies.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro.

Reconstituted High-Density Lipoprotein Attenuates Cholesterol Crystal-Induced Inflammatory Responses by Reducing Complement Activation.

Chronic inflammation of the arterial wall is a key element in the development of atherosclerosis, and cholesterol crystals (CC) that accumulate in plaques are associated with initiation and progression of the disease. We recently revealed a link between the complement system and CC-induced inflammasome caspase-1 activation, showing that the complement system is a key trigger in CC-induced inflammation. HDL exhibits cardioprotective and anti-inflammatory properties thought to explain its inverse correlation to cardiovascular risk. In this study, we sought to determine the effect of reconstituted HDL (rHDL) on CC-induced inflammation in a human whole blood model. rHDL bound to CC and inhibited the CC-induced complement activation as measured by soluble terminal C5b-9 formation and C3c deposition on the CC surface. rHDL attenuated the amount of CC-induced complement receptor 3 (CD11b/CD18) expression on monocytes and granulocytes, as well as reactive oxygen species generation. Moreover, addition of CC to whole blood resulted in release of proinflammatory cytokines that were inhibited by rHDL. Our results support and extend the notion that CC are potent triggers of inflammation, and that rHDL may have a beneficial role in controlling the CC-induced inflammatory responses by inhibiting complement deposition on the crystals.

TNT003, a Novel Anti-C1s Monoclonal Antibody, Inhibits Anti-HLA Antibody Induced Complement Deposition.

PURPOSE: To determine whether the anti-C1s antibody, TNT003, inhibits complement deposition induced by anti-HLA Class I and II antibodies (Ab). METHODS: HLA reference and transplant patient sera were used as sources of anti-HLA Ab. Complement deposition was evaluated using C1qScreen (OneLambda) and C3d solid phase assays (Immucor/Lifecodes). Briefly, single antigen beads were coated with anti HLA Ab, incubated with normal human serum complement, and C1q or C3d production/deposition were detected by monoclonal PE conjugates and quantified by Luminex analysis. For in vitro studies, either HLA-typed primary human aortic endothelial cells (HAECs) or EBV-immortalized B cells (B cells) were incubated with sera containing cognate anti-HLA Ab and normal human complement serum, and deposition of complement proteins was detected by flow cytometry using monoclonal antibodies. Complement split products produced during deposition reactions were measured by ELISA. TNT003 (True North Therapeutics Inc, CA) was titrated into these experiments in a concentration-dependent manner (0-50 μg/mL). RESULTS: The anti-C1s monoclonal Ab, TNT003, inhibited complement deposition in both solid phase and in vitro assays, and complement split product formation during in vitro assays. TNT003 reduced both anti-HLA Class I and II Ab induced C3d but not C1q deposition on single antigen beads in a concentration-dependent manner. In vitro, increasing concentrations of TNT003 were inversely proportional to levels of complement deposition on ECs that had been treated with anti-HLA Ab; complement split product production was also diminished with TNT003 treatment. Similar results were seen using B cells as a source of HLA, as TNT003 addition to the complement deposition reaction decreased levels of complement proteins on the cell surface, as well as split products in the supernatant of these reactions. CONCLUSIONS: We demonstrate here that TNT003, a novel anti-C1s monoclonal Ab, is capable of inhibiting complement activation, deposition, and split product formation induced by anti-HLA Class I and II Ab in both solid phase and cell based in vitro assays. These data suggest TNT003 may be useful in reducing DSA-induced complement-mediated damage in transplant patients experiencing AMR. DISCLOSURES:Thomas, K.: Grant/Research Support, True North Therapeutics Inc. Valenzuela, N.: Grant/Research Support, True North Therapeutics Inc. Parry, G.: Employee, True North Therapeutics Inc. Panicker, S.: Employee, True North Therapeutics Inc. Reed, E.: Grant/Research Support, Pfizer, True North Therapeutics Inc, Novartis, Immucor.

Pneumococcal Surface Protein A Inhibits Complement Deposition on the Pneumococcal Surface by Competing with the Binding of C-Reactive Protein to Cell-Surface Phosphocholine

In the presence of normal serum, complement component C3 is deposited on pneumococci primarily via the classical pathway. Pneumococcal surface protein A (PspA), a major virulence factor of pneumococci, effectively inhibits C3 deposition. PspA's C terminus has a choline-binding domain that anchors PspA to the phosphocholine (PC) moieties on the pneumococcal surface. C-reactive protein (CRP), another important host defense molecule, also binds to PC, and CRP binding to pneumococci enhances complement C3 deposition through the classical pathway. Using flow cytometry of PspA(+) and PspA(-) strains, we observed that the absence of PspA led to exposure of PC, enhanced the surface binding of CRP, and increased the deposition of C3. Moreover, when the PspA(-) mutant was incubated with a pneumococcal eluate containing native PspA, there was decreased deposition of CRP and C3 on the pneumococcal surface compared with incubation with an eluate from a PspA(-) strain. This inhibition was not observed when a recombinant PspA fragment, which lacks the choline-binding region of PspA, was added to the PspA(-) mutant. Also, there was much greater C3 deposition onto the PspA(-) pneumococcus when exposed to normal mouse serum from wild-type mice as compared with that from CRP knockout mice. Furthermore, when CRP knockout mouse serum was replenished with CRP, there was a dose-dependent increase in C3 deposition. The combined data reveal a novel mechanism of complement inhibition by a bacterial protein: inhibition of CRP surface binding and, thus, diminution of CRP-mediated complement deposition.

The Absence of PspA or Presence of Antibody to PspA Facilitates the Complement-Dependent Phagocytosis of PneumococciIn Vitro

Pneumococcal surface protein A (PspA) is a surface molecule on pneumococci that is required for full virulence in mouse models of infection. PspA has been reported to inhibit complement deposition on the pneumococcal surface. It has been assumed that this decreased complement deposition results in the inefficient phagocytosis of wild-type pneumococci. However, an effect of PspA on phagocytosis had not been shown. Our present studies demonstrated that a loss of PspA by capsular type 3 strains WU2 and A66.1 led to enhanced complement-dependent phagocytosis of the pneumococci by the mouse macrophage cell line J774A.1. This observation was made using human complement as well as mouse complement. Since this enhanced phagocytosis could be blocked by antibody to complement receptor CR3 on J774A.1, it was concluded that PspA's effect on phagocytosis was due to its effect on the amount of deposited complement, which in turn helped opsonize the pneumococci for phagocytosis. Since these studies included new independent mutants lacking PspA, the results provide solid confirmation of the previously reported effects of PspA on pneumococcal virulence and complement deposition. Finally, we showed that antibody to PspA, which is also known to enhance complement deposition, also enhances the phagocytosis of pneumococci in a largely complement-dependent manner.

Protective effect of scFv-DAF fusion protein on the complement attack to acetylcholine receptor: A possible option for treatment of myasthenia gravis

Autoantibody-induced complement activation, which causes disruption of the postsynaptic membrane, is recognized as a key pathogenic factor in myasthenia gravis (MG). Therefore, specific targeting of complement inhibitors to the site of complement activation is a potential therapeutic strategy for treatment of MG. We assessed expression of single-chain antibody fragment-decay accelerating factor (scFv-DAF), comprising a single-chain fragment scFv1956 based on the rat complement inhibitor DAF in prokaryotic systems, and studied its inhibitory effect on complement deposition in vitro. The recombinant conjugate scFv-DAF completely retained the wild-type binding activity of scFv1956 to AChR and inhibited complement activation of DAF in vitro. We found that scFv-DAF could bind specifically to TE671 cells, and it is significantly more potent at inhibiting complement deposition than the untargeted parent molecule DAF. scFv-DAF may be a candidate for in vivo protection of the AChR in MG.

Soluble CD59 Expressed from an Adenovirus In Vivo Is a Potent Inhibitor of Complement Deposition on Murine Liver Vascular Endothelium

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited.

CspA-Mediated Binding of Human Factor H Inhibits Complement Deposition and Confers Serum Resistance in Borrelia burgdorferi

Borrelia burgdorferi has developed efficient mechanisms for evading the innate immune response during mammalian infection and has been shown to be resistant to the complement-mediated bactericidal activity of human serum. It is well recognized that B. burgdorferi expresses multiple lipoproteins on its surface that bind the human complement inhibitors factor H and factor H-like protein 1 (FH/FHL-1). The binding of FH/FHL-1 on the surface of B. burgdorferi is thought to enhance its ability to evade serum-mediated killing during the acute phase of infection. One of the key B. burgdorferi FH/FHL-1 binding proteins identified thus far was designated CspA. While it is known that CspA binds FH/FHL-1, it is unclear how the interaction between CspA and FH/FHL-1 specifically enhances serum resistance. To better understand how CspA mediates serum resistance in B. burgdorferi, we inactivated cspA in a virulent strain of B. burgdorferi. An affinity ligand blot immunoassay and indirect immunofluorescence revealed that the CspA mutant does not efficiently bind human FH to its surface. Consistent with the lack of FH binding, the CspA mutant was also highly sensitive to killing by human serum. Additionally, the deposition of complement components C3, C6, and C5b-9 was enhanced on the surface of the CspA mutant compared to that of the wild-type strain. The combined data lead us to conclude that the CspA-mediated binding of human FH confers serum resistance by directly inhibiting complement deposition on the surface of B. burgdorferi.

Pneumococcal histidine triad proteins are regulated by the Zn 2+ ‐dependent repressor AdcR and inhibit complement deposition through the recruitment of complement factor H

The pneumococcal histidine triad (Pht) proteins are a recently recognized family of surface proteins, comprising 4 members: PhtA, PhtB, PhtD, and PhtE. They are being promoted for inclusion in a multicomponent pneumococcal protein vaccine currently under development, but to date, their biological functions and their relative contributions to pathogenesis have not been clarified. In this study, the involvement of these proteins in pneumococcal virulence was investigated in murine models of sepsis and pneumonia by using defined, nonpolar mutants of the respective genes in Streptococcus pneumoniae D39. In either challenge model, mutagenesis of all 4 genes was required to completely abolish virulence relative to the wild-type, suggesting significant functional redundancy among Pht proteins. The in vivo expression of pht genes was significantly up-regulated in the nasopharynx and lungs compared with blood. We provide unequivocal molecular evidence for Zn(2+)-dependent, AdcR-mediated, regulation of pht gene expression by real-time reverse transcriptase-polymerase chain reaction, Western blotting, and electrophoretic mobility-shift assays. We also present the first direct evidence for the biological function of this protein family by demonstrating that Pht proteins are required for inhibition of complement deposition on the pneumococcal surface through the recruitment of complement factor H.

Capsule does not block antibody binding to PspA, a surface virulence protein of Streptococcus pneumoniae

Of the proteins on the surface of Streptococcus pneumoniae, one of those best able to elicit protection against pneumococcal infection is pneumococcal surface protein A (PspA). Although this protein is attached to the membrane molecule, lipoteichoic acid, which is well beneath the capsule, PspA's ability to inhibit complement deposition and killing by apolactoferrin, suggests that it must have surface exposure. This study provides quantitative data showing that the capsular polysaccharide on types 2 and 3 pneumococci provides little or no masking ability of antibodies to bind PspA. Capsule was even observed to enhance, rather than inhibit the binding of two protective monoclonal antibodies to their epitopes on cell surface PspA. These results with antibodies to PspA are in contrast to binding by antibodies to the phosphocholine (PC) epitope of the lipoteichoic and teichoic acids. The binding of antibody to PC was largely, but not completely, blocked by capsular polysaccharide.

Investigation of antibody-coated liposomes as a new treatment for immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disease that is mediated by anti-platelet antibodies. It is believed that anti-platelet antibody-opsonized platelets are eliminated through Fcγ receptor-mediated and complement-mediated phagocytosis by macrophages of the reticuloendothelial system (RES). Polyclonal pooled immunoglobulin with high titer for the D-antigen of erythrocytes (i.e., anti-D) has been successfully used to ameliorate ITP. Based on the pathogenesis of ITP and based on the successful application of anti-D for the treatment of ITP, we hypothesized that antibody-coated liposomes may be used to inhibit Fcγ receptor-mediated and complement-mediated phagocytosis, thereby increasing platelet counts in ITP. To test this hypothesis, we have developed a liposome preparation that is coated with a model monoclonal IgG1 antibody. Antibody-coated liposomes were found to inhibit complement deposition and macrophage phagocytosis in vitro. Furthermore, antibody-coated liposomes were also found to attenuate thrombocytopenia in a rat model of ITP, in a dose-dependent manner. The results suggest that antibody-coated liposomes may be used as ‘decoy particles’ to competitively inhibit the destruction of antibody-coated platelets; thus, antibody-coated liposomes may have value in the treatment of ITP.

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.

Invasive Streptococcus pneumoniae isolates from Argentinian children: serotypes, families of pneumococcal surface protein A (PspA) and genetic diversity.

PspA is an antigenically variable virulence factor of Streptococcus pneumoniae that inhibits complement deposition and is a potential candidate for human vaccines. Of 64 published strains 96% are in PspA families 1 and 2; optimal protection is family-specific. Effective development of a PspA-containing vaccine requires more information about the PspA family of strains in parts of the world where the vaccine is most needed. In these studies we observed that of 149 isolates (of 19 capsular types) from Argentina, 54.4% were family 1, 41.6% were family 2 and 4.0% expressed both family 1 and family 2 PspAs. Box typing revealed the Argentinian strains to be from at least 10 clonally related groups.

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants.

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.