Complement can neutralize HIV-1 plasma virus by a C5-independent mechanism.

A previous study showed a portion of HIV-1 plasma virus was lysed by the addition of exogenous human AB+ seronegative complement. The current study was performed to determine whether infectious plasma virus was inactivated by complement. Incubation of plasma virus with AB+-seronegative serum resulted in substantial decreases in infectious titers, demonstrating that infectious plasma virus is susceptible to complement-mediated inactivation. Although complement also induced some lysis of plasma virus samples, virus was neutralized to a significantly higher degree, suggesting neutralization did not occur solely by lysis. Additionally, C5-deficient complement substantially neutralized virus, indicating coating of virus by early complement components was an important mechanism of neutralization. A portion of some freshly isolated plasma virus samples bound to complement receptor 2 in the absence of exogenous complement, indicating that early complement components bound virus in vivo. Furthermore, plasma virus samples that had less C3 deposited on their surface in vivo had higher infectious titers than samples with a larger fraction with surface C3. These findings suggest that complement can neutralize HIV-1 plasma virus in vivo by coating with complement proteins. This is the first study to provide evidence that coating by complement leads to functional inactivation of a virus in vivo.

Mechanisms of resistance of HIV-1 primary isolates to complement-mediated lysis.

Previous studies suggested that HIV-1 primary isolates (PI) were resistant to complement-mediated lysis (CML), while virus produced in certain T cell lines and virus taken directly from the plasma of HIV+ persons were both susceptible to CML. The purpose of this study was to investigate the mechanism(s) of PI resistance. PI were resistant to CML using pooled seropositive serum as an antibody source. Additionally, PI obtained from two patients at several times over 2 years were resistant to CML using autologous antibody. PI were also resistant to CML induced by monoclonal antibodies which neutralize a broad range of PI. Resistance to CML was associated with low binding of antibody to PI but was not due to low gp120 levels. Cell-line-derived virus and PI were equally sensitive to CML induced by antibody to host-cell proteins, suggesting that PBMC do not contribute properties to virions which make them more physically resistant to CML in general but that PI resistance is restricted to CML induced by antiviral antibody. These studies show that PI are resistant to CML mediated by various antiviral antibodies and indicate that low binding of antibody to virus is an important factor contributing to resistance.

A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells.

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp701ucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp701ucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.

Detection of HLA-DR associated with monocytotropic, primary, and plasma isolates of human immunodeficiency virus type 1.

This study determined whether HLA-DR was incorporated into human immunodeficiency virus type 1 produced in vivo or by primary cultured cells. HLA-DR was associated with virions from primary isolates, macrophage cultures, and blood plasma. These results represent the first demonstration of major histocompatibility complex molecules associated with an in vivo source of virus.

Susceptibility of HIV-1 plasma virus to complement-mediated lysis. Evidence for a role in clearance of virus in vivo.

This study was undertaken to directly assess the susceptibility of HIV-1 plasma virus to C-mediated lysis. Plasma from HIV-infected individuals was collected and ultracentrifuged over 20% sucrose to isolate virions from plasma components including anticoagulants, which inhibit C activity. Treatment with C alone in the absence of exogenously added Ab caused lysis of virus from all patients (n = 18) (range 14 to 86%). This lysis occurred via the classical C pathway and was not due to cross-reactive Abs in the C source. Protein A bound a fraction of isolated plasma virus and this binding was blocked by purified human Ig suggesting that anti-HIV Abs bound to plasma virus could be responsible for inducing C activation. A portion of virus bound to CR2 on cells in the absence of exogenously added C indicating that virus activated C in vivo. C levels from six of six patients were determined to be sufficient to lead to lysis of virus in vivo. Since plasma virus appeared more sensitive to C than primary isolates, isolated virus was evaluated for the presence of C control proteins. While primary isolate virions contained CD46, CD55, and CD59, only CD59 was detected on plasma virus. The results of this study strongly suggest that C is activated by a portion of plasma virus in vivo due to the binding of Ab. The resultant opsonization plus subsequent lysis may be important routes of clearance and destruction of plasma virus in infected persons.

Anti-cellular antibodies in sera from vaccinated macaques can induce complement-mediated virolysis of human immunodeficiency virus and simian immunodeficiency virus.

Previous studies show that immunization of macaques with preparations of either human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) that has been produced in human cells can induce antibodies against both viral antigens and human cellular antigens. This is due to the fact that certain host cell antigens are carried along with the virus during the purification process. The current series of experiments were performed to determine whether these anti-cellular antibodies can activate complement and whether the resultant complement activation could lead to virolysis of either HIV or SIV. Sera from macaques immunized with SIV or HIV (produced in the H9 human cell line) contained anti-cellular antibodies as determined by flow cytometry. Antibodies in these sera were capable of activating complement on uninfected human cells. Sera from the HIV-immunized macaques induced complement-mediated virolysis of both HIV and SIV. Similarly, sera from SIV-immunized macaques induced complement-mediated virolysis of both SIV and HIV. These results suggested that anti-cellular antibody in the sera could induce complement-mediated virolysis of either virus. To investigate this further, sera was absorbed with uninfected cells, which removed all of the virolytic activity for the heterologous virus. These in vitro studies indicate that complement activation can be initiated by anti-human cell antibodies, and that this activation can result in the destruction either HIV or SIV. This unusual antiviral mechanism may account for some portion of the resistance of human cell-immunized macaques to human cell-produced SIV that has been recently reported.

Monoclonal antibody to the type II Fc receptor for human IgG blocks potentiation of monocyte and neutrophil IgG-induced respiratory burst activation by aggregated C-reactive protein.

The acute phase protein, CRP, when heat-aggregated (Agg-CRP), binds to human monocytes and neutrophils and potentiates the respiratory burst stimulated by heat-aggregated IgG (Agg-IgG). Earlier data from our laboratory and others have indicated that CRP binds to phagocytic cells at membrane sites associated with IgG Fc receptors. The present study utilized monoclonal antibodies (MAb) to determine whether the Agg-CRP potentiation of oxidative metabolism could be linked to activation through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Preincubation of monocytes with MAb 32.2, which recognizes an Fc gamma RI epitope distinct from its IgG binding site, had only a minimal (20%) inhibitory effect on Agg-IgG-induced luminol chemiluminescence (CL) and exerted no significant effect on its enhancement by Agg-CRP. MAb 10.1, which blocks IgG binding to Fc gamma RI, reduced Agg-IgG-induced monocyte CL by 40%, but did not alter the Agg-CRP-mediated enhancement. In contrast, exposure to MAb IV.3, which binds to Fc gamma RII on monocytes and neutrophils and blocks IgG binding to this receptor, resulted in a greater than 70%, inhibition of Agg-IgG-induced CL and also significantly suppressed the enhancement by Agg-CRP. MAb Leu-11b, which reacts with Fc gamma RIII on neutrophils, reduced Agg-IgG-induced CL by 70% but did not suppress the Agg-CRP potentiation. Preincubation of monocytes and neutrophils with anti-Leu-M1, anti-CR1, or anti-CR3 failed to block Agg-IgG-induced CL or its enhancement by Agg-CRP. Although the potentiating effect of Agg-CRP on Agg-IgG-elicited CL was blocked by MAb IV.3, this antibody failed to reduce binding of Agg-CRP to either monocytes or neutrophils. These results indicate that, although Agg-CRP does not bind to phagocytic cells at the IgG-binding determinant of Fc gamma RII, it alters Agg-IgG-induced cell activation through this receptor.

Complement activation by human monoclonal antibodies to human immunodeficiency virus.

It has been shown that the incubation of human immunodeficiency virus (HIV) with polyclonal antibodies from HIV-infected persons and complement results in complement-mediated neutralization due, at least in part, to virolysis. The current study was performed to determine whether any of a panel of 16 human monoclonal antibodies to HIV could activate complement and, if so, which determinants of the HIV envelope could serve as targets for antibody-dependent complement-mediated effects. Human monoclonal antibodies directed to the third variable region (V3 region) of HIVMN gp120 induced C3 deposition on infected cells and virolysis of free virus. Antibodies to two other sites on HIVMN gp120 and two sites on gp41 induced few or no complement-mediated effects. Similarly, only anti-V3 antibodies efficiently caused complement-mediated effects on the HIVIIIB isolate. In general, the level of C3 deposition on infected cells paralleled the relative level of bound monoclonal antibodies. As expected, pooled polyclonal antibodies from infected persons were much more efficient than monoclonal antibodies inducing C3 deposition per unit of bound immunoglobulin. Treatment of virus or infected cells with soluble CD4 resulted in increases in anti-gp41 antibody-mediated virolysis and C3 deposition but decreases in anti-V3 antibody-mediated virolysis and C3 deposition. In general, virolysis of HIV was more sensitive as an indicator of complement-mediated effects than infected-cell surface C3 deposition, suggesting the absence of or reduced expression of functional complement control proteins on the surface of free virus. Thus, this study shows that human monoclonal antibodies to the V3 region of gp120 are most efficient in causing virolysis of free virus and C3 deposition on infected cells. Elution of gp120 with soluble CD4 exposes epitopes on gp41 that can also bind antibody, resulting in virolysis and C3 deposition. These findings establish a serologically defined model system for the further study of the interaction of complement and HIV.

C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils.

The acute phase protein, C-reactive protein (CRP), when heat-aggregated (Agg-CRP), potentiates immunoglobulin G (IgG) Fc receptor-mediated luminol-enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of monocytes and neutrophils to Agg-CRP, superoxide anion release was measured by cytochrome c reduction. In addition, the extracellular release of hydrogen peroxide was distinguished from hydrogen peroxide generation using a phenol red oxidation assay. Finally, a flow cytometric determination of dichlorofluorescein (DCFH) oxidation was employed as an index of intracellular peroxide production. Although Agg-CRP alone did not stimulate hydrogen peroxide generation by either monocytes or neutrophils, it significantly enhanced hydrogen peroxide generation in response to heat-aggregated IgG (Agg-IgG). In contrast, Agg-CRP did not enhance the extracellular release of either hydrogen peroxide or superoxide anion from Agg-IgG-stimulated cells. The capacity of Agg-CRP to enhance selectively intracellular oxidative product generation was confirmed when measuring DCFH oxidation in Agg-IgG-stimulated cells. To evaluate whether this selective enhancement of intracellular oxidative events could be attributed, at least in part, to a scavenging effect of Agg-CRP, a cell-free oxygen radical-generating system was employed. Agg-CRP did not significantly diminish the lucigenin-amplified CL response induced by the xanthine/xanthine oxidase reaction. These results indicate that although Agg-CRP enhances the intracellular generation of reactive oxygen intermediates by monocytes and neutrophils, extracellular release of those products is not influenced by cell interaction with Agg-CRP. It is tempting to speculate that CRP can selectively boost the microbicidal activities of monocytes and neutrophils within an inflammatory site by amplifying the intracellular generation of reactive oxygen products without increasing damage to surrounding normal tissues.

Human immunodeficiency virus (HIV)-infected cells and free virus directly activate the classical complement pathway in rabbit, mouse and guinea-pig sera; activation results in virus neutralization by virolysis.

Since animal models of human immunodeficiency virus (HIV) infection are being used increasingly in determining various aspects of virus/host interaction and as models for virus expression, it will be important to assess any significant differences in anti-viral immune responses between animals and humans. Previous studies have shown that incubation of HIV with non-immune sera from several animal species results in virus neutralization, and that rabbit serum can lyse HIV-infected cells. The objectives of the current study were to evaluate the animal complement pathway(s) activated by HIV and HIV-infected cells and determine the mechanism by which complement could mediate viral neutralization. Incubation of HIV-infected cells with mouse, guinea-pig or rabbit sera resulted in cell-surface deposition of C3 fragments. Deposition of C3 fragments did not occur either in the presence of C4-deficient guinea-pig serum or in the absence of Ca2+, indicating that activation by infected cells occurred via the classical pathway. Neutralization of free virus was also mediated by the classical pathway since C4-deficient guinea-pig serum and Ca(2+)-chelated sera lacked activity. Serum treatment of virus resulted in release of HIV reverse transcriptase (RT), suggesting that neutralization occurred by C5b-9-mediated virolysis. RT was also released from simian immunodeficiency virus by animal complement. Antibodies in animal sera were not responsible for the classical pathway activation by free virus or HIV-infected cells. These results define several substantial differences between animal and human complement reactivity with HIV which could significantly affect the ability of HIV to replicate in animals, and which need to be considered in the assessment of animal models of HIV infection.

Direct binding of complement component C1q to human immunodeficiency virus (HIV) and human T lymphotrophic virus-I (HTLV-I) coinfected cells.

Previous studies have shown that coinfection of the human T lymphotrophic virus type I (HTLV-I) chronically infected cell line MT4 with human immunodeficiency virus type 1 (HIV-1) results in cells which spontaneously activate complement via the classical pathway. This complement activation was antibody independent, yet required C2, suggesting either direct C1, C4, or C2 activation. Because some animal retroviruses have been shown to bind human C1q directly, the present study investigated the possible direct binding of C1q by HIV coinfected MT4 cells. Coinfected cells bound both C1q present in serum and highly purified C1q. Binding of C1q resulted in formation of active C1 on the cell surface, which could in turn activate complement as shown by C4 consumption. The C1q binding was not HIV-isolate specific since infection of MT4 cells with any of three diverse isolates all induced C1q binding. Purified collagen-like region (CLR) and globular region (GR) fragments of C1q both bound to coinfected cells, suggesting a mechanism of binding by C1q similar to that of fibronectin-C1q binding. However, culture of coinfected cells in serum-free (fibronectin-free) medium did not reduce C1q binding. A second HTLV-I chronically infected line, SLB-1, also displayed increased binding of C1q after HIV infection. The H9 cell line, which is not HTLV-I infected, did not bind C1q after HIV infection. These results suggest that a retrovirus protein expressed by coinfected cells directly binds C1q resulting in classical complement activation. This type of activation may have profound biological effects in persons coinfected with HIV-1 and HTLV-I.

Neutralization of human immunodeficiency virus type 1 by complement occurs by viral lysis.

The ability of complement to inactivate human immunodeficiency virus (HIV) in the presence of specific antibody was evaluated. HIV was treated with complement and/or antibody, and then its titer was determined on the CD4+ H9 cell line. While complement alone had no effect on the HIV titer, complement plus subneutralizing levels of antibody resulted in titer reductions. Complement sources deficient in membrane attack component C5 or C8 did not inactivate antibody-treated HIV, suggesting that neutralization occurred via lysis. This possibility was investigated by assessing release of reverse transcriptase (RT) from the virion. Antibody plus complement, but neither reagent alone, released RT from HIV in a dose-dependent manner. Release of RT did not occur with C5- or C8-deficient sera, also indicating a requirement for membrane attack components. These studies show that complement can neutralize HIV via the classical complement pathway and that this neutralization occurs via C5b-9-mediated viral lysis. Thus, complement may play a major role in resistance to disease by lysing HIV and preventing infection of Fc- and complement receptor-positive cells, as well as CD4+ cells.

Activation of complement on the surface of cells infected by human immunodeficiency virus.

Cells were infected with HIV-1 and tested for C activation using a flow cytometric assay for bound C3 fragments. HIV-infected H9 cells bound increased levels of C3 using normal human serum as a C source only after cells were first incubated with serum containing anti-HIV antibody. Uninfected H9 cells or infected cells incubated with HIV-antibody negative sera did not bind C3. Although C3 bound quickly and was maximal within 10 min, modulation of bound C3 was slow with about 50% loss after 4 h. C3 binding required specific anti-HIV antibody, was blocked by EGTA, and did not occur in C2-deficient serum suggesting that binding was via the classical pathway. The HTLV-1-infected MT-4 cell line also bound high levels of C3 after coinfection with HIV. C3 binding in HIV-infected MT4 cells was also mediated via the classical pathway because it was not observed in Mg-EGTA chelated or C2-deficient sera. However, this classical pathway activation appeared to be antibody independent because it was also detected in HIV-antibody negative serum and a-gamma-globulinemic serum. This indicates that coinfection with HTLV-1 and HIV-1 can produce novel C activating conditions. No cytotoxic effect of human C for antibody-treated HIV-infected cells was observed in a chromium release assay. However, rabbit C was cytotoxic for HIV-infected cells in the absence of anti-HIV antibodies. Our results suggest that C can be activated in vivo by infected cells via specific anti-HIV antibody. The resultant C3 deposition on infected cells could have profound effects on interaction with CR-bearing cells.