Neutralization of X4- and R5-tropic HIV-1 NL4-3 variants by HOCl-modified serum albumins

Abstract

Background: Myeloperoxidase (MPO), an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl) from H2O2 and chloride, which is released into body fluids. Besides its direct microbicidal activity, HOCl can react with amino acid residues and HOCl-modified proteins can be detected in vivo.

Findings: This report is based on binding studies of HOCl-modified serum albumins to HIV-1 gp120 and three different neutralization assays using infectious virus. The binding studies were carried out by surface plasmon resonance spectroscopy and by standard ELISA techniques. Virus neutralization assays were carried out using HIV-1 NL4-3 virus and recombinant strains with CXCR4 and CCR5 coreceptor usage. Viral infection was monitored by a standard p24 or X-gal staining assay. Our data demonstrate that HOCl-modified mouse-, bovine- and human serum albumins all bind to the HIV-1 NL4-3 gp120 (LAV) glycoprotein in contrast to non-modified albumin. Binding of HOCl-modified albumin to gp120 correlated to the blockade of CD4 as well as that of V3 loop specific monoclonal antibody binding. In neutralization experiments, HOCl-modified serum albumins inhibited replication and syncytium formation of the X4- and R5-tropic NL4-3 isolates in a dose dependent manner.

Conclusions: Our data indicate that HOCl-modified serum albumin veils the binding site for CD4 and the V3 loop on gp120. Such masking of the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when HOCl-modified serum albumin is applied, for example, as a topical microbicide.

Figure 1

Effect of HOCl on HSA and the ability to inhibit syncytium formation. To test the transformation effect of HOCl on HSA, mHSA samples were produced, which were generated using different HOCl:HSA ratios. The different mHSA preparations were tested for the inhibition of gp120/gp41-induced syncytia. HeLa-P4 cells were transfected with pSVATGrev plasmid expressing the NL4-3 (LAV) env. Syncytia were visualized by a standard staining procedure (Hemacolor^®, Merck) and the nuclei of fused cells were counted. Shown are the averages of 6 wells.

Figure 2

Binding of HOCl-modified serum albumins to gp120. (a-f) Binding of HSA, BSA, MSA and HOCl-modified albumins mHSA, mBSA, mMSA (protein:HOCl molar ratio 1:1000) to LAV-gp120 was tested by surface plasmon resonance spectroscopy (Biacore 1000, GE Healthcare). Purified LAV gp120 (10 μg/ml, Protein Sciences Corporation) was covalently linked using EDC/NHC to a dextran coated, CH-activated sensor chip (CM5, GE Healthcare). After blocking with ethanolamine the biosensor was washed three times with 30 μl 100 mM NaOH and 30 μl 100 mM HCl (flow rate 5 μl/min). Proteins were tested for binding at 1, 5, 10, 25 and 50 μg/ml at a flow rate of 5 μl/min. (g). Binding of mHSA, mBSA and mMSA (values marked by the black arrow in Figure 1a, 1c and 1e) was compared to the binding of CD4, and gp120-specific antibodies to a LAV gp120 coated CM5 biosensor. ADP429: anti-serum, derived from HIV-1 SF2 gp120-immunized sheep (AIDS reagent project, NIBSC, UK). EVA3047: monoclonal antibody, derived from IRIQRGPGRAFTIGC-peptide immunized mice (AIDS reagent project, NIBSC, UK).

Figure 3

Inhibition of CD4 and antibody binding to gp120. Binding of CD4 or antibody, in the presence of mHSA, mBSA and mMSA, to LAV gp120 was tested by ELISA. ELISA plates were coated with LAV gp120 (1 μg/ml, Protein Sciences Corporation) over night. After gp120 binding, the wells were coated using BSA (20 mg/ml). Modified serum albumin was added to the wells and (a) CD4 (1 μg/ml, Protein Sciences Corporation) or (b) antibody diluted in phosphate buffered saline (PBS) pH 7.5, was added 30 min later. After 1 hour, the plates were washed 5-times with PBS. Binding of CD4 to gp120 was carried out at various mSA concentrations. Gp120-bound CD4 was detected by a CD4 specific mouse monoclonal antibody (NCL-CD4-1F6, Novocastra) followed by a staining with an anti-mouse HRP-antibody conjugate (BioRad). Shown are the averages of triplicate wells. Binding of V2- and V3-loop-specific monoclonal antibodies (mab) was carried out in the presence of 10 μg/ml of mHSA. Gp120-bound antibody was detected using an anti-mouse-HRP-antibody conjugate (BioRad). Antibodies were designated according to the repository reference of the Centralized Facility for AIDS Reagents. In brackets: originators antibody designation. All measurements were carried out in triplicates. White symbols: V2 loop specific mab. Black symbols: V3 loop-specific mab.

Figure 4

Inhibition of NL4-3 infection. (a) Effects of HOCl-modified serum albumins (molar ratio 1:1000) on cell growth. HeLa-P4 cells were cultured in the presence of MTT and various concentrations of mHSA, mBSA and mMSA (white symbols, 1, 5, 20 and 50 μg per ml; Black symbols, non-modified serum albumins). Cells were seeded at 10⁴ cells per well in 96-well plates in triplicate. After adding the yellow tetrazolium salt MTT, metabolically active cells produced purple formazan. The amount of formazan was measured at 530 nm and is a correlate for cell viability and proliferation. (b) NL4-3 neutralization by HOCl-modified serum albumins (molar ratio 1:1000). HeLa-P4 cells were infected with 1000 TCID of the HIV-1 laboratory strain NL4-3. Shown are the averages of triplicate wells. Infection was monitored 5 days after infection by a standard p24 antigen ELISA.

Figure 5

Inhibition of NL4-3 and gp120-induced syncytium formation. (a-c) Inhibition of virus induced syncytium formation. GHOST-CXCR4 cells were infected with 1000 TCID of the HIV laboratory isolates NL4-3. Infection was inhibited by adding mHSA (final concentration 50 μg/ml). Cell cultures were monitored 5 days after infection by visualization of induced syncytia and cell layer destruction after formalin fixation followed by a standard staining procedure (Hemacolor^®, Merck). (d-f) To study inhibition of syncytia formation following transfection with pSVATGrev, cells were transfected with the expression plasmid and mHSA was added 30 min later. Syncytia were monitored after 28 h by standard phase contrast microscopy.

Figure 6

Inhibition of X4, X4R5 and R5 tropic NL4-3 isolates by mHSA. (a) V3 loop amino acid sequences of NL4-3 mutants. NNT, recognition site for N-glycosylation. (b) TZM-bl cells were infected with virus supernatant representing an infectious dose of 500 foci/96-well. Infection was inhibited by adding mHSA (molar HSA:HOCl ratio 1:1000) to a final concentration of 1, 10, 50, 100 and 150 μg per ml of culture medium. Viral entry was monitored 2 days after infection by X-gal staining and counting of blue foci. Shown are the averages of triplicate wells. White symbols: X4 tropic viruses. Black symbols X4R5 (NL952) and R5 tropic viruses. The NL4-3 isolates were constructed by an exchange of the NL4-3 V3 loop against V3 loop sequences from primary isolates PI 952, PI-930, PI-918 and PI-991.