Complement-mediated binding of naturally glycosylated and glycosylation-modified human immunodeficiency virus type 1 to human CR2 (CD21).

Particulate glycoproteins lacking sialic acid, such as desialylated enveloped viruses, readily activate complement through the alternative pathway. Human immunodeficiency virus type 1 (HIV-1) contains two heavily glycosylated and partially sialylated envelope glycoproteins: a surface gp120 and a transmembrane gp41. The abilities of naturally glycosylated HIV-1 and glycosylation-modified HIV-1 to interact with the complement system were examined with a biological assay which measured the binding of whole virus particles to cells expressing CR2 (CD21), the complement receptor found naturally in abundance on follicular dendritic cells and immature B cells. HIV-1 IIIB was synthesized in the presence or absence of the mannosidase II inhibitor, swainsonine, to give rise to high-mannose-type, nonsialylated, nonfucosylated carbohydrate moieties. The virus also was treated with neuraminidase or endo-beta-galactosidase to remove terminal sialic acids. An enzyme immunoassay specific for HIV-1 p24 core protein was used to quantitate the amount of virus bound to cell surfaces. Virus particles incubated with 1:3-diluted, fresh HIV-1-negative human serum as a source of complement readily bound to MT-2 (CD4+ CR2+) and Raji-3 (CD4- CR2+) cells but not to CEM (CD4+ CR2-) cells, suggesting that the virus bound to CR2 independently of CD4. Compared with heat-inactivated or C3-deficient sera, fresh complement increased binding by as much as 62 times for naturally glycosylated virus, and 5 times more than this for glycosylation-modified virus. Similar observations were made with freshly isolated, non-mitogen-stimulated peripheral blood mononuclear cells. Additional evidence that HIV-1 bound to CR2 independently of CD4 was provided by the fact that binding was blocked by monoclonal antibody OKB7 (anti-CR2) but not by OKT4a (anti-CD4). Also, the virus bound to transfected K562 cells (CD4-) which expressed recombinant human CR2 but did not bind to untransfected K562 cells. Results obtained with complement component-deficient sera indicated that binding required the alternative complement pathway. Raji-3 and transfected K562 cells could not be infected with HIV-1 in the presence of complement, suggesting that utilization of CR2 as a receptor in the absence of CD4 does not allow virus entry. The demonstration of CR2 as a receptor for HIV-1 in the presence of complement, together with the ability to enhance binding by desialylation, provides new insights into mechanisms of HIV-1-induced immunity and immunopathogenesis.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

Complement mediates the binding of HIV to erythrocytes

A fraction of HIV is associated with erythrocytes even when the virus becomes undetectable in plasma under antiretroviral therapy. The aim of the present work was to further characterize this association in vitro. We developed an in vitro model to study the factors involved in the adherence of HIV-1 to erythrocytes. Radiolabeled HIV-1 (HIV) and preformed HIV-1/anti-HIV immune complexes (HIV-IC) were opsonized in various human sera, purified using sucrose density gradient ultracentrifugation, and incubated with human erythrocytes. We observed that, when opsonized in normal human serum, not only HIV-IC, but also HIV, bound to erythrocytes, although the adherence of HIV was lower than that of HIV-IC. The adherence was abolished when the complement system was blocked, but was maintained in hypogammaglobulinemic sera. Complement-deficient sera indicated that both pathways of complement were important for optimal adherence. No adherence was seen in C1q-deficient serum, and the adherence of HIV was reduced when the alternative pathway was blocked using anti-factor D Abs. The adherence could be inhibited by an mAb against complement receptor 1. At supraphysiological concentrations, purified C1q mediated the binding of a small fraction of HIV and HIV-IC to erythrocytes. In conclusion, HIV-IC bound to erythrocytes as other types of IC do when exposed to complement. Of particular interest was that HIV alone bound also to erythrocytes in a complement/complement receptor 1-dependent manner. Thus, erythrocytes may not only deliver HIV-IC to organs susceptible to infection, but free HIV as well. This may play a crucial role in the progression of the primary infection.     

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

Neutralization of vesicular stomatitis virus (VSV) by human complement requires a natural IgM antibody present in human serum

Neutralization of VSV by human serum ws previously shown to involve C1, C2, C3, and C4 of the classical complement (C) pathway. All normal human sera tested were equivalently active in this regard. However, purified C1, C2, C3, and C4 were unable to mediate VSV neutralization. In the present studies an additional factor required for C-mediated neutralization was isolated from normal human serum and identified as a natural IgM antibody specific for a viral encoded antigen. Purified IgM bound to the virus and formed a complex that activated component C1. Normal serum concentrations of purified IgM, C1, C2, C3, C4 neutralized VSV to the same extent as normal serum. Purified IgM did not neutralize VSV alone or in conjunction with C1, C2, and C4. Inclusion of C3 resulted in full neutralization and C3b binding to the virus was demonstrated. Thus, normal human serum contains a natural antibody of the IgM class that is directed toward a viral antigen. The antibody facilitates neutralization by forming an immune complex that activates C1 and thus efficiently initiates the classical pathway at the viral surface. Neutralization occurs with C3b deposition on the viral envelope and probably results from a blanket of C protein that interferes with viral attachment to susceptible cells.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.     

Brucella abortus conjugated with a gp120 or V3 loop peptide derived from human immunodeficiency virus (HIV) type 1 induces neutralizing anti-HIV antibodies, and the V3-B. abortus conjugate is effective even after CD4+ T-cell depletion.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.     

A complement-resistant HeLa cell line (T638) is blocked at the step of C3 deposition

A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-susceptible HeLa cells in anti-beta 2-microglobulin (b2m) antiserum and complement. The T638 line maintained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mechanism of resistance of T638 cells to complement-mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CH50 activity completely from normal human serum; cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cells bound more 3H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was fourfold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 3H-C3. The T1/2 for decay of C142-mediated binding of 3H-C3 on HeLa was 3.9 min, whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3 convertase.     

Activation of the alternative complement pathway by isolated human glomerular basement membrane

The capacity of isolated human glomerular basement membrane (GBM) to initiate surface activation of the human alternative complement pathway was defined by the deposition of C3b under circumstances in which the classical complement pathway was inoperative. The deposition of C3b from normal or C2-deficient serum was time- and magnesium-dependent, implying a role for the alternative pathway. Normal human serum rendered deficient in D did not sustain C3b deposition until its reconstitution with D, indicating an absolute requirement for a protein unique to the alternative pathway and essential to the cleavage activation of the C3 amplification convertase of that pathway. The capacity of the excess control proteins H and I to prevent C3b deposition onto GBM incubated in C2-deficient serum provided further evidence for the direct activation of the alternative pathway in this system. The use of radiolabeled monoclonal antibody to localize the deposited C3b afforded specificity and quantitation of about 100 ng of C3b/mg of GBM. Immunohistochemical analysis with a monoclonal antibody to detect C3b demonstrated its deposition to be confined to the epithelial surface of the GBM.     

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.     

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

Antibodies to discontinuous or conformationally sensitive epitopes on the gp120 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans.

We have used an indirect-capture enzyme-linked immunosorbent assay to quantitate the reactivity of sera from human immunodeficiency virus type 1 (HIV-1)-infected humans with native recombinant gp120 (HIV-1 IIIB or SF-2) or with the gp120 molecule (IIIB or SF-2) denatured by being boiled in the presence of dithiothreitol with or without sodium dodecyl sulfate. Denaturation of IIIB gp120 reduced the titers of sera from randomly selected donors by at least 100-fold, suggesting that the majority of cross-reactive anti-gp120 antibodies present are directed against discontinuous or otherwise conformationally sensitive epitopes. When SF-2 gp120 was used, four of eight serum samples reacted significantly with the denatured protein, albeit with ca. 3- to 50-fold reductions in titer. Only those sera reacting with denatured SF-2 gp120 bound significantly to solid-phase-adsorbed SF-2 V3 loop peptide, and none bound to IIIB V3 loop peptide. Almost all antibody binding to reduced SF-2 gp120 was blocked by preincubation with the SF-2 V3 loop peptide, as was about 50% of the binding to native SF-2 gp120. When sera from a laboratory worker or a chimpanzee infected with IIIB were tested, the pattern of reactivity was reversed, i.e., there was significant binding to reduced IIIB gp120, but not to reduced SF-2 gp120. Binding of these sera to reduced IIIB gp120 was 1 to 10% that to native IIIB gp120 and was substantially decreased by preincubation with IIIB (but not SF-2) V3 loop peptide. To analyze which discontinuous or conformational epitopes were predominant in HIV-1-positive sera, we prebound monoclonal antibodies (MAbs) to IIIB gp120 and then added alkaline phosphatase-labelled HIV-1-positive sera. MAbs (such as 15e) that recognize discontinuous epitopes and compete directly with CD4 reduced HIV-1-positive sera binding by about 50%, whereas neutralizing MAbs to the C4, V2, and V3 domains of gp120 were either not inhibitory or only weakly so. Thus, antibodies to the discontinuous CD4-binding site on gp120 are prevalent in HIV-1-positive sera, antibodies to linear epitopes are less common, most of the antibodies to linear epitopes are directed against the V3 region, and most cross-reactive antibodies are directed against discontinuous epitopes, including regions involved in CD4 binding.     

Susceptibility of Helicobacter pylori to the Bactericidal Activity of Human Serum

Background. Human serum represents an important barrier to the entry of most mucosal organisms into tissues and to the systemic circulation. If at all present, Helicobacter pylori within gastric tissue is rare, and bacteremia for this organism has been described only once.
Methods. To assess the susceptibility of H. pylori to the bactericidal activity present in normal human serum (NHS), we examined 13 H. pylori isolates. To assess the contributions of the classical and alternative complement pathways to killing, we added either C2-deficient or factor B-deficient serum, respectively, to heat-inactivated NHS. Also we assessed the ability of the strains to bind ¹²⁵I-C3.
Results. After incubation for 60 minutes at 37°C, all 13 H. pylori strains were killed by NHS; heating to 56°C for 30 minutes ablated killing, indicating complement dependence for this phenomenon. In the absence of an antibody source, there was no killing when either an alternative or classical complement pathway source was used. Adding B-deficient serum to heat-inactivated normal human serum did not restore killing, but adding C2-deficient serum permitted partial killing. All of the 13 strains bound ¹²⁵I-C3. Although the kinetics varied from strain to strain, C3 bound was significantly correlated ( r = 0.61, p = 0.03) with serum susceptibility.
Conclusions. H. pylori are susceptible to complement, alternative pathway activation appears critical, and C3 binding is a major locus of variability.     

Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation.

Living Schistosoma mansoni schistosomula incubated with normal chicken, guinea pig, human, and monkey sera were killed after 4 hr contact at 37 degrees C. The following data indicate that this action is dependent on the activation of the alternative complement pathway (AP): a) the inactivity of RB, RD, and zymosan-treated serum against schistosomula; b) the partial activity of RD restored in FD; c) the full effect of the C4-deficient guinea pig, C2-deficient human, and the agammaglobulinemic human sera; d) the consumption of both the AP and FB after the incubation of NHS with schistosomula; e) the detection of C3d breakdown product during the contact of the C2-deficient human serum with these young parasites. Killing by serum was decreased as the immature schistosomes developed and was completely absent against 4-day-old lung schistosomula (LS). In other experiments, it was demonstrated that schistosomula, in the presence of IgG, were able to initiate complement activation also through the classical pathway (CP). However, the CP does not appear to play a role in the schistosomulicidal activity of complement. The in vivo relevance of these observations is considered.     

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.