MH, MF, HFG, and AT designed the study. MH, MF, VvW, and AT analyzed the data. RW and HFG enrolled patients. MH, MF, AM, HK, RW, VvW, HFG, and AT contributed to writing the paper. BM developed the assay for the determination of GP41 antibody titers, determined all the antibody titers (anti-gp41, anti-gp120, and anti-p24), and analyzed these data. AM provided an experimental contribution to assays. HK performed virus isolations and neutralization assays and determined plasma IgG titers. BN performed RNA extractions and real-time PCRs. RW contributed to the design of the clinical studies and collection of patients' data. HFG designed and conducted the clinical studies forming the base for this laboratory work and was significantly involved in the overall design of this investigation.
The authors declare that no competing interests exist.
To explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus.
Sera from two groups of patients—25 with acute HIV-1 infection and 31 with chronic infection—were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env) proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies.
Collectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.
Antibody-mediated complement lysis of HIV virions develops rapidly and is effective already early in the course of HIV infection.
If untreated, most people who become infected with the human immunodeficiency virus (HIV) eventually develop acquired immunodeficiency syndrome (AIDS). Over time, HIV infects and kills their CD4 T lymphocytes—immune system cells that stimulate B lymphocytes to make antibodies (proteins that recognize and destroy infectious agents) and that help CD8 T lymphocytes to kill cells that contain viruses and bacteria. The loss of CD4 T lymphocytes—a central player in “adaptive immunity”—leaves patients very susceptible to infections. However, the immune system does not die quietly. It does its best to fight HIV infection by mounting a cell-mediated immune response in which T lymphocytes attack HIV-infected cells. It also mounts a “humoral” immune response in which antibodies that recognize HIV are made. Some of these are neutralizing antibodies, which prevent HIV entering its host cells and replicating. Other antibodies may limit viral spread by inducing destruction of the virus. One way they can do this is by activating another part of the immune system called the complement system, which can break open and kill viruses (this is known as antibody-mediated complement lysis). In addition, antibodies and complement can coat the HIV virus particles so that phagocytes (for instance macrophages—yet another type of immune system cell) engulf and destroy the virus.
The role that humoral immunity plays in fighting HIV infection is complex and poorly understood. In particular, it is not clear whether the complement system helps to stop the spread of HIV or whether it inadvertently helps it to spread by facilitating its entry into host cells. It is important to understand as much as possible about the humoral immune response to HIV infection so that vaccines can be designed to provide maximum protection against HIV. In this study, the researchers have investigated whether antibody-mediated complement lysis controls the amount of virus in the blood of patients infected with HIV.
The researchers collected plasma (the liquid part of blood that contains circulating antibodies) from patients recently infected with HIV (acute infection) and patients who had been infected for some time (chronically infected). They also isolated HIV from each of the patients—so-called autologous virus. They then used a sensitive molecular biology assay to test each plasma sample for its ability to lyse the autologous virus (and also a standard virus) when supplied with complement from a healthy donor. Most of the plasma samples were able to lyse HIV, although the samples taken from chronically infected patients generally caused more lysis than those from acutely infected patients. In the chronically infected patients, the level of lysis induced was not related to the amount of virus in the patients' blood (viremia). However, plasma taken from acutely infected patients with higher viral loads was less active in the lysis assay than plasma taken from patients with lower viral loads. Finally, the researchers showed that the levels of antibodies in the various plasma samples to the two envelope proteins of HIV correlated strongly with the ability of each sample to lyse the standard virus and that these antibodies were mainly non-neutralizing antibodies.
By showing that antibody-mediated complement lysis of HIV in the laboratory is inversely related to the patients' viral loads during acute infection, these findings suggest (but do not prove) that antibody-mediated complement lysis of HIV contributes to the control of viremia early in HIV infections. But, the importance of this form of humoral immunity in combating HIV infections remains uncertain, since complement has the potential to enhance as well as block viral spread. Further work is needed to unravel which of these effects is dominant in patients and to characterize fully the antibodies that activate complement. Nevertheless, the results of this study suggest that complement-activating antibodies should be considered in future attempts to design an effective HIV vaccine.
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The humoral immune response to HIV-1 infection is elicited early in infection and is generally vigorous at later disease stages, but its efficacy and modes in controlling viremia in vivo have not yet been completely unraveled. While numerous studies suggest that neutralizing antibodies may impact viral replication in vivo [
The complement system is a key component of innate immune defense, and it provides a link to the adaptive immune response [
The role of complement in HIV pathogenesis appears to be multifaceted [
Opposing these observations, several reports have suggested that complement lysis of HIV-1 is limited in vivo by several host cell-derived complement control proteins incorporated by the HIV-1 virions [
In the present study we sought to investigate the question of whether antibody-mediated complement lysis of HIV-1 contributes to virus containment in vivo and, if so, to quantify the relative contribution of this defense mechanism at different disease stages.
The study included 25 acutely infected and 31 chronically infected patients (
Patient blood was sampled in Vacutainer tubes containing EDTA (BD [http://www.bd.com]), and plasma was collected within 6 h and frozen in 1 ml aliquots at −75 °C. Plasma was heat inactivated (1 h at 56 °C) to destroy complement activity and centrifuged at 500
A mix of sera from one to five healthy donors, stored at −75 °C, was used as source of complement.
HIV-1 virus stock (25 μl) was incubated with 20 μl of patient plasma (final dilution 1:5), 50 μl of complement (NHS final dilution 1:2), and 1 μl of RNase A (Qiagen, Valencia, California, United States) in RPMI 1640 (BioWhittaker [
The extraction efficiency was controlled by adding and quantifying synthetic murine prion protein (PrP) mRNA (7,866 copies/μl) as an internal standard. In each assay, samples were tested in triplicate. Complement-mediated lysis activity was expressed as the percentage of lysed HIV-1 RNA copies compared to control plasma treatment. A mixture of plasma from 1–5 HIV-1-negative donors was used as negative control (no lysis activity, 0% value).
HIV-1 virions were quantified using primers either to Gag or to the 5′ end of HIV-RNA. For detection, dual-labeled fluorescent probes with a fluorescein (FAM) moiety at the 5′ ends and a tetramethylrhodamine (TAMRA) moiety at the 3′ end were used. HIV-Gag was measured using primers skcc1b [
Plasma IgG titers to recombinant gp120 from the JR-FL strain (kindly provided by W. Olson, Progenics, Tarrytown, New York, United States), recombinant gp41 (amino acids 541–682 of the HxB2 strain [Viral Therapeutics, Ithaca, New York, United States]) and recombinant p24 (IIIB [Aalto BioReagents, Dublin, Ireland]) were determined by ELISA as described [
Plasma IgG titers to recombinant gp41 were determined as described for the other two antigens using plates coated with 0.1 μg of gp41 per well (amino acids 541 to 682 of the HxB2 strain; Viral Therapeutics). Maximal binding to gp41 was defined using the antibody 2F5 (kindly provided by H. Katinger) as a reference. Detection of bound antibody and calculation of midpoint titers were done as described [
IgG was depleted from patient plasma using Protein G Sepharose beads (Amersham Pharmacia [
Neutralization activity of patient plasma against replication competent autologous primary virus isolates was evaluated on peripheral blood mononuclear cells as described [
Neutralization activity of patient plasma against the heterologous virus isolate JR-FL was evaluated on TZM-bl cells (National Institutes of Health AIDS Research and Reference Reagent Program) using JR-FL Env pseudotyped virus as described [
Inhibitory activity of patient plasma in the presence of active or heat-inactivated complement was evaluated using JR-FL Env pseudotyped virus on TZM-bl cells [
Data analyses were performed using Prism version 4.03 for Windows (GraphPad Software, San Diego, California, United States) and Stata SE/9.2 for Windows (Stata Corporation, College Station, Texas, United States). The normality assumption was checked using the D'Agostino and Pearson omnibus normality test and had to be rejected for most variables (unpublished data). Hence, nonparametric methods were employed for testing of group differences (Mann-Whitney U test and Wilcoxon signed-rank test for unpaired and paired testing, respectively). Correlation analysis was performed using Spearman's rank correlation. All tests of significance were two-tailed and the level of significance was set at 0.05. P values reported in the main text and figures refer to values obtained after singular testing. Since multiple testing was performed caution must be taken in evaluating significance. In sum, 47 tests of significance were performed in our study, thus our level of significance was set at
The primary intent of this study was to evaluate the influence of antibody- and complement-mediated lysis on viremia control in vivo. To this end we utilized an assay strategy that allows reliable and sensitive quantification of virus lysis by complement. In order to probe complement lysis activity under in vivo-relevant conditions, virus preparations used in our study were derived from infected primary peripheral blood mononuclear cells, because these cells are known to incorporate high numbers of complement control proteins, rendering these viruses less susceptible to complement lysis than those produced on immortalized cell lines [
Patient Characteristics, Complement Lysis Activity, Antibody Titers, and Neutralization Titers
We further validated our assay and ensured that freeze thaw-cycles by themselves do not disrupt virions. As previously described, we found that neither the single freeze-thaw cycle used in our assay nor repeated cycles (2–4) led to disintegration of the virus in the absence of patient sera (
(A) Primary HIV-1 virions were incubated with autologous plasma and complement and freeze-thawed once, and viral RNA was digested by RNase and DNase. After inactivation of RNase and DNase by protease and addition of an internal standard (PrP RNA), RNA was extracted and quantified by real-time PCR.
(B) HIV-1 virions sensitive to antibody-mediated complement lysis were disrupted, making viral RNA accessible for degradation.
(C) Viral RNA of complement lysis-resistant virions remained intact. RNA was extracted and could be quantified by real-time PCR.
To determine if the observed virolysis activity depends on antibodies, we depleted plasma from two patients of IgG using Protein G Sepharose beads prior to incubation with virus and complement (
To explore the impact of antibody-mediated complement virus lysis at different disease stages we measured patient plasma complement virolysis activity against autologous virus in a previously described cohort of 25 acutely and 31 chronically infected patients (
(A) Plasma from HIV-positive individuals specifically lyses HIV in presence of active complement. Virus isolate JR-FL was incubated with complement (+), without complement (−), or with inactivated complement (Ci) either in the absence of plasma (−) or with plasma from uninfected individuals (NHP) or from patient 106. One of three independent experiments is shown.
(B) Up to four freeze-thaw cycles do not destroy intact HIV-1 virions. Virus JR-FL was incubated in the presence of active complement with medium (no plasma), with plasma from uninfected persons (NHP), or with plasma from patient 106. Reaction mixtures were subjected to the indicated number of freeze-thaw cycles and the effect on virus disintegration was measured. One of two independent experiments is shown.
(C) IgG depletion reduces lysis activity. Plasma of uninfected individuals (NHP) and of patients 117 and 113 were depleted of IgG with Protein G Sepharose beads (darkened bars) and lysis activity was compared to untreated plasma (open bars). Error bars indicate standard deviation of triplicate measurements. Groups were compared using Mann-Whitney U test.
Virus input ranged from 4.8 ×103 to 6.3 × 107 viral RNA copies. In two patients with very high viral load (patients 018 and 022), virus detected in the plasma contributed over 1% to the total amount of HIV-1 copies measured in our assay. Therefore, patient plasma and heat-inactivated complement were used as negative control in these cases.
Autologous plasma induced complement lysis of the respective virus in the majority of infected patients (
Antibody responses to HIV-1, particularly during early disease stages, are thought to be predominantly strain specific. To investigate whether the antibodies that mediate complement lysis activity are specific for the autologous virus isolate or if cross-reactivity with heterologous isolates exists, we evaluated the capacity of our panel of patient plasmas to mediate lysis of the heterologous primary virus strain JR-FL. All plasma samples derived from chronically infected individuals, and 19 of 25 samples derived from acutely infected individuals mediated lysis of the virus strain JR-FL (
In a further analysis, autologous and heterologous lysis activities were assessed within each group and then separately in the combined cohort. In general, plasma lysis activity against heterologous and autologous viruses did not differ in the acutely or the chronically infected group. The analysis of the entire patient cohort also revealed no differences in lysis activity (
Complement-mediated lysis activity against autologous (A) and heterologous (JR-FL) (B) virus was compared between acutely (blue triangles) and chronically (green circles) infected individuals. HIV-specific lysis activity is present at acute and chronic diseases stages but generally higher during chronic infection. Crosses denote uninfected plasma controls. Groups were compared using Mann-Whitney U test.
In the course of HIV-1 infection, commonly a strong, high-titered antibody response to the structural viral proteins (Env and Gag) is elicited [
In order to probe the impact of anti-Env antibody responses on viral lysis activity, we first analyzed the interdependency between lysis activity against JR-FL and antibody titers to the gp120 protein of this strain. We found a strong correlation between anti-gp120 titers and lysis activity when we analyzed the entire patient cohort (rho = 0.87,
Group Comparisons of Measured Parameters
Correlation Analyses
Antibody titers against the autologous virus strains could not be determined. Due to the heterogeneity of virus isolates in our panel and the resulting variable sequence divergence in the recombinant proteins used to determine antibody titers, it was unlikely that we would see the same degree of association between autologous lysis activity and binding activity to the recombinant proteins. Nevertheless, a trend of a positive nonsignificant association between gp120 binding titers and lysis activity was observed in acutely infected individuals (rho = 0.47,
Our cross-sectional analysis suggested that complement lysis activity is mediated by anti-Env responses and may increase when the antibody response broadens. To investigate the development of the lysis activity more closely, we monitored antibody responses and autologous complement lysis activity over extended time periods in plasma of six acutely HIV infected patients (
(A) Correlation analysis of anti-gp120 and anti-gp41 antibody titers in plasma samples from the acute and chronic patient groups shows that titers to the envelope glycoprotein correlate tightly independently of disease stage.
(B–D) Correlation analyses of heterologous lysis (JR-FL) with anti-gp120 (B) or anti-gp41 (C) revealed positive correlations, whereas anti-p24 (D) antibody titers did not associate with heterologous lysis. This suggests that lysis activity is driven by envelope-specific antibodies.
Blue triangles denote acutely, green circles denote chronically infected patients. Spearman's rank correlation coefficient (rho) and
Lysis activity against autologous virus (red circles) of plasma from six acutely HIV-1-infected patients was measured longitudinally and plotted against anti-gp120 titers (orange triangles), anti-gp41 titers (blue squares), and viral load (asterisks). At the first data point, patients were treatment-naïve and acutely HIV infected. All patients (except patient 022) subsequently went on antiretroviral therapy for the indicated time periods. Time point 0 was assigned to the date of treatment interruption and the remaining time points were calculated according to this time point. (A) Patient 022 (treatment-naïve), (B) patient 015, (C) patient 003, (D) patient 016, (E) patient 026, and (F) patient 002.
Complement lysis activity of non-neutralizing and neutralizing patient plasmas was compared in the acute and the chronic infection cohorts. Autologous neutralization (A and B) and heterologous (JR-FL) (C and D) neutralization are shown, with blue triangles denoting acutely, green circles chronically infected patients. Each data point represents the mean of two or three independent experiments with plasma from the same patient. Groups were compared using Mann-Whitney U test.
As expected, titers of antibody to gp120 and gp41 increased steadily in all patients, which was paralleled by an increase in lysis activity. In the five patients who underwent treatment interruption it is evident that along with viral load levels, antibody responses and lysis activity rose. A direct impact on in vivo viral load levels cannot be easily investigated in this setting, since upon rebound it takes several weeks to months before set points of viral load and immune responses are reached. Collectively, our observation strongly suggests that early antibody responses against the viral Env proteins gp41 and gp120 mediate lysis activity against the autologous virus strain.
Neutralizing antibodies directed against the autologous HIV strain can appear during the acute infection phase [
Only six (24%) of the plasmas from the 25 acutely infected patients and 14 (45%) from the group of 31 chronic patients showed measurable autologous neutralization activity (NT90 > 40) (
Although none of the patient plasmas from the acute group showed neutralizing activity against the heterologous virus strain JR-FL (
Since our virus lysis assay measures complement destruction under nonphysiological conditions (freeze-thaw cycle and RNA digestion) we investigated whether virion lysis occurs also under natural conditions and leads to a reduction of viral infectivity. To circumvent nonspecific inhibitory or enhancing effects of human plasma in our in vitro assay, we chose assay conditions in which controls contained the corresponding concentration of normal human plasma and complement. To be able to compare effects of antibodies in the absence of active complement (neutralization) and inhibition induced in the presence of active complement (neutralization and complement lysis), we chose plasma dilutions that allowed simultaneous evaluation of both effects. Thus, patient plasmas from the acute group, all of which had marginal neutralization activity against the isolate JR-FL, were studied at a dilution of 1:40; chronic patients, whose plasma generally had higher neutralization activity, were studied at a dilution of 1:200. Each patient group was assessed separately, which allowed us to use two different plasma dilutions. The latter was necessary, because otherwise neutralization activity would have dominated the readout in the chronic group. In the majority of patients the presence of active complement increased the inhibitory effect of the patient plasma (
Both HIV-1-specific T cell activity and—somewhat delayed—humoral immune responses develop early in HIV infection. The initial rise in cytotoxic T lymphocytes responses is often associated with a decline in viremia shortly after infection [
Inhibitory activity of patient plasma against the heterologous virus JR-FL was measured in presence of active (C+) or heat-inactivated (Ci) complement on TZM-bl cells. Blue triangles denote acutely, green circles chronically infected patients. Data points are means of two independent experiments with plasma from the same patient. Differences in inhibition between inactivated and active complement within the subgroups were compared using Wilcoxon signed-rank test. The results demonstrate that complement increases the inhibitory activity of HIV specific antibodies in vitro.
We found that lysis activity against autologous plasma inversely correlated with the in vivo viral load (RNA copies/ml of plasma) in the acute group (rho = −0.72,
In recent years substantial effort has been put into investigating the humoral immune response to HIV-1. While neutralizing antibodies are considered a correlate of protection against HIV-1 and a necessary component of vaccine-induced immune responses, the role of effector mechanisms mediated by anti-HIV antibodies in immune control remains largely unclear. In the present study we investigated the efficacy of the humoral immune response elicited during acute and chronic disease stages in inducing complement-dependent lysis of HIV virions. Evidence obtained through a novel complement virion lysis assay suggests that antibody-mediated complement lysis in the plasma of HIV-1-infected individuals has been underestimated in the past. Previously used virolysis assays mostly relied on the measurement of reverse transcriptase [
In vitro-determined autologous virolysis activity was correlated with in vivo HIV-1 RNA copies measured per milliliter of plasma in the acutely infected group (A) and the chronically infected group (B). Correlations were evaluated using Spearman's rank correlation. Our data demonstrate that increased antibody-mediated complement lysis coincides with lower viral loads in the acute phase. No evidence was found for a similar correlation in the chronically infected group.
We found that, in most patients, antibodies are elicited very early after infection (< 3 mo) that induce complement-mediated lysis of the autologous virus and thus could contribute to viremia control during the acute phase of HIV-1 infection. The latter is signified in our study by a tight inverse correlation between lysis activity and in vivo-measured viral loads. Our results corroborate the findings by Aasa-Chapman and coworkers, who recently reported complement lysis activity in sera of some acutely infected individuals [
In support of the differences between acute and chronic group we found that, particularly during the acute phase, complement lysis appeared to be predominantly mediated by non-neutralizing antibodies. Lysis activity against the heterologous strain JR-FL was observed despite the absence of neutralizing antibodies against this virus in acutely infected patients. Likewise, lysis and neutralization activity against the autologous viruses showed no interdependency. Although in the chronic cohort higher lysis activity was found in patients that harbored neutralizing activity, we observed no direct relationship of these reactivities against the autologous virus, and only a minor influence in the heterologous system. Nevertheless, we cannot exclude the possibility that neutralizing antibodies at concentrations lower than the detection limit of the neutralization assay are present and contribute to virolysis activity.
The role of complement in HIV pathogenesis has been a matter of debate for many years. While several reports have proposed that complement-dependent virus lysis occurs in vivo [
The factors that direct complement action to either lysis or enhancement have not been fully defined. While our study demonstrates inhibitory effects of complement and antibody in early HIV infection, complement-dependent enhancement of HIV infection of complement receptor-bearing cells likely occurs as well in vivo. Complement concentration may vary at different disease stages [
Moreover, complement concentrations in tissue are only in the range of 10%–20% of the levels in serum [
It is currently also not known whether antibodies that mediate complement lysis and enhancement are directed to the same epitopes. Nevertheless, it seems feasible that their reactivities overlap to some extent. Our data indicate that if enhancing activity impacts on virus replication in vivo, it appears not to be a direct consequence of high antibody titers, as we observed no notable interdependency between binding antibody titers to HIV and viral loads. We demonstrated, however, that also under culture conditions, complement in concert with HIV-1-specific antibodies reduces viral infectivity, which further supports a role for virus lysis in vivo.
We determined in our study that complement lysis activity is mediated by anti-Env antibodies, as demonstrated by the tight correlation between lysis activity and antibody titers to gp120 and gp41. While neutralizing antibodies are known to recognize native viral Env oligomer epitopes that are involved in receptor binding and fusion, complement-activating antibodies are not limited to these sites. Additionally, non-neutralizing antibodies binding to the oligomer, or antibodies reacting with gp120 monomers or with epitopes on gp41 that are exposed after shedding of gp120, could potentially activate complement if bound to the virion in sufficient densities.
A more detailed characterization of the antibodies mediating lysis activity will be particularly important if complement-activating antibodies are to be considered a component of effective vaccines.
Our finding that HIV-1 is susceptible to lysis mediated by specific antibodies in patient sera and complement is in agreement with a previous study by Sullivan and coworkers, who showed that virus derived from patient plasma can be lysed in the presence of complement due to virion-bound antibodies that activate the complement system [
Several recent reports have emphasized that complement activation boosts humoral and cellular immune responses [
While our study demonstrates that presence and magnitude of autologous antibody-mediated complement lysis of HIV-1 coincide with increased viremia control during the acute infection phase, direct associations cannot be formally proven. Immune functions and the timing of their appearance during the course of early HIV-1 are intertwined, and it is therefore difficult to ascertain direct relationships. Activities of cytotoxic T cells, neutralizing antibodies, antibodies that mediate antibody-dependent cellular toxicity, opsonization, aggregation, phagocytosis and—as our current report suggests—antibodies that mediate virus lysis via activation of the complement system will impact on viral spread in vivo. Total or neutralizing antibody titers or CTL activity, however, have not been proven to reliably predict viremia levels in the past [
The varying amounts of RNA input into the autologous assays were correlated with measured autologous virolysis activities. Correlation was evaluated using Spearman's rank correlation (
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We thank our patients for their commitment, and Roland Hafner, Barbara Hasse, Urs Karrer, Rolf Oberholzer, Elisabeth Presterl, Christine Schneider, and Christina Grube for excellent patient care.
neutralization titer