In the present study, we demonstrated that HOCl-modified serum albumins can bind to LAV gp120, and that HOCl-modified serum albumins possess the ability to efficiently neutralize entry of the NL4-3 laboratory strain. Since the 5'-half of NL4-3 was constructed from NY5 and the 3'-half from LAV the NL4-3 external envelope is identical to the commercial LAV gp120 used in our SPR binding studies.
Binding between the LAV gp120 and our inhibitor was observed in a SPR assay with gp120 covalently linked to a biosensor chip. Binding to gp120 was only monitored with HOCl-modified serum albumin and non-modified serum albumin showed no binding activity. As shown before , mHSA binding is also specific to the West Nile virus domain III and does bind to similar domains of Yellow Fever and Dengue-2 virus, respectively. Since mHSA also binds to WNV envelope, we suggest that the binding of HOCl-modified serum albumin to HIV-1 or WNV envelope is neither a highly epitope specific event nor does it mirror a fundamental, yet undiscovered, molecular step of the WNV or HIV-1 entry process. Such non-specific binding events are common in many other pathogen-host interactions. For example malaria parasites use negatively charged chondroitin-sulfate-proteoglycan (CSPG) structures to attach to the membrane surface of their host cells . For HIV-1, heparan-sulfate-proteoglycan (HSPG) is such an attachment factor  and heparan sulphates as well as other polyanions are efficient inhibitors of HIV-1 infection [21, 22]. Our current hypothesis is that pathogens which coat their core particle with the membrane of the host cell need a simple mechanism to attach to cells possessing an identical membrane structure. This membrane-membrane contact must be mediated by the external envelope glycoprotein complex present in the viral membrane and might be a first non-specific binding event to initiate the next step, binding to specific receptors.
Masking the envelope complex to prevent membrane attachment seems to be a simple mechanism to prevent viral entry. The blockade of CD4 and V3-loop antibody binding by mHSA supports this hypothesis. The masking by mHSA completely inhibited rCD4 binding and partially inhibited the V3-loop antibody binding, indicating that a large and important proportion of the gp120 molecule is masked by mHSA. The surface of gp120 is per se covered by complex carbohydrate structures, which represent more than 55% of the gp120 molecular weight. Especially the elimination of the carbohydrate structure g15 within the V3 loop plays an important role in viral entry and these mutants showed higher infectivity. Due to higher infectivity, these viruses can escape from neutralizing antibodies present in human sera . An interesting advantage of mHSA is the ability to neutralize these antibody resistant viruses. A more efficient neutralization of such viruses by mHSA might be the result of the lack of N-glycan g15 that renders the V3 loop more exposed. Such an unmasked V3 loop seems to be a better binding target for HOCl-modified albumins since the binding of V3-loop-specific neutralizing antibodies was blocked by mHSA. Masking the CD4 and V3 loop area by mHSA was also efficient for the blockade of CCR5-specific infection, but 3 times higher levels were necessary compared to the neutralization of CXCR4-viruses. Thus, V3-driven selection for higher infectivity of CXCR4 viruses or the switch from CCR5- to CXCR4-usage is not beneficial for promoting escape from mHSA neutralization. Blocking both coreceptor pathways is an important step to block HIV-1 transmission. In an evolving epidemic, CXCR4-tropism is present in high frequency among all HIV-1 subtypes except subtype C [24, 25]. The high frequency of CXCR4-tropism, 86% that was found in subtype A and CRF02_AG in patient samples from West Africa, demonstrates that a topical microbicide has to inhibit CXCR4-viruses frequently present in HIV-1 infected individuals. On the other hand, cervico-vaginal tissue preferentially supports the productive infection by CCR5-tropic viruses . Thus, a microbicide has to block both coreceptor pathways to prevent HIV-1 sexual transmission. The HOCl-modified serum albumins, when applied as a topical microbicide, can be applied in an adequate concentration (>200 μg/ml) that is high enough to block both viruses completely.
In our experiments we added HOCl-modified serum albumin directly into cell culture media. Viral infection assays in cell culture have the advantage that cell culture ingredients such as (i) antioxidants present in fetal calf serum or (ii) free amino acids like methionine or cysteine present in cell culture medium at 0.2 M are present during the test period. In addition, the mHSA samples produced with low amounts of HOCl showed no inhibitory effect indicating that potential traces of taurine or HOCl are not contributing to the inhibitory effect. We also tested our inhibitor in three different neutralization assays using three different cell types (HeLa, GHOST and TZM-bl) which are well established as host cells, widely used in the HIV literature. Since virus is neutralized under all these different cell culture conditions, all the cell culture ingredients will not suppress the antiviral activity of mHSA. Preincubation of cells with mHSA also had no suppressive effect, showing that mHSA is not non-specifically adsorbed by the cells or their different membrane components. Moreover, the normal growth of other viruses like yellow fever and dengue-2 virus in cell cultures, over a period of up to 9 days, containing mHSA  indicates that mHSA has no unspecific overall neutralizing activity or toxic activity that inhibits cellular growth and more especially viral production. We have also tested mHSA in other sensitive cell culture systems (Plasmodium falciparum growth in erythrocytes, Lassa-, Marburg-, SARS virus growth) over a time period of 14 days (data not shown). In all these cell culture experiments we observed no inhibitory effect on pathogen growth indicating that mHSA in not a cellular toxin per se.
The data implicate that mHSA, or a well defined structure thereof, might be applicable as a drug against viral infection, and that the inoculation of mHSA into infected individuals might overcome adaptive immune tolerance. As was shown in animals, the active immunization of rats with Freund's adjuvant together with a high dose of chlorinated autoantigen induced an immune response against the HOCl-modified protein . Induction of autoantibody might be a risk factor in the direct application of HOCl-modified proteins into body fluids but seems to be no major problem when applied as a topical microbicide. On the other hand, serum albumin is an important carrier for drugs and enzymes. In particular the transport of the enzyme MPO across the endothelial barrier depends on a specific interaction between HSA and MPO . The HSA-binding epitope on MPO was identified as a short linear region (aa 425-454), causing high-affinity binding to HSA. The very close proximity of HSA and MPO implicates that HSA might be highly susceptible for MPO-dependent modifications. This also suggests that MPO-modified serum albumin is a common self antigen and therefore might be tolerated by the immune system.
It is known that HOCl is a powerful oxidant and modifications of protein structure and function are well documented. In published studies, HOCl-induced modifications were analyzed after treatment of peptides or proteins with a 0.5-25-fold molar excess of HOCl [18, 29–31]. As shown by Vossmann et al.  as well as in the present study, an antiviral activity of serum albumins was observed only after treatment of serum albumin with HOCl concentrations above the molar ratio of 1:100. Using lower HOCl concentration, e.g. 1:10 or 1:50 we were unable to detect any antiviral activity in our HIV-1 infection assays. This observation is important and demonstrates that HOCl modifications, which occur under low HOCl conditions [18, 29–31] might be totally different to those induced with higher HOCl concentrations as shown here. Another minor aspect is that we have modified albumin at concentrations ≤ 1 mg protein/ml. When serum albumin was treated with HOCl at higher concentrations, as described in the literature (10 mg/ml) , we observed no inhibitory activity in our viral infection and syncytium assays (data not shown). Thus, based on our current knowledge, the antiviral activity of serum albumins was induced in diluted aqueous solution and only with a >100-fold molar excess of HOCl.
The modification of proteins by HOCl seems to be a highly complex reaction. Data describing this process are available mainly based on the structure analysis of modified amino acids or peptides at low HOCl concentration. In a report by Salavej et al. , HSA was treated with HOCl at a 25-fold molar excess, similar to the procedure by Pattison et al. . In their analysis, oxygenation was detected for Trp238, Met147, Met353, and Met572, but no chlorination of any of the HSA residues was detected. Using HOBr, Salavej and coworkers detected the incorporation of one or two bromines at Tyr425, but no chlorination of any HSA residues was detected in HOCl treated samples . However, the treatment of amino acid residues with HOCl leads to the documented side chain modifications, but HOCl-modification seems not to be limited to these side chain products. In the context of a linear peptide or protein HOCl-modified side chains undergo intra- or inter-molecular cross linking. Thus, in the context of a complex protein and probably under conditions with excessive concentrations of HOCl, the process is pushed towards the development of complex protein conformations the nature of which is still unknown. One final product of HOCl-treatment seems to be the generation of stable antigenic epitopes on human serum albumin.
Monoclonal antibodies against HOCl-induced epitopes, have been produced. These monoclonal antibodies were mainly raised against oxLDL. One of these monoclonal antibodies was able to bind to the HOCl-induced epitopes on HSA and based on this antibody (clone 2D10G9) HOCl-modified proteins can be detected in human tissue [13–15, 29]. It was suggested that the epitope recognized by this monoclonal antibody is of a complex conformational type, but almost nothing is known about the nature of the 2D10G9 epitope. The fact that antibodies can be induced by immunization and HOCl-induced epitopes can easily be detected in human tissue  indicates that these HOCl-induced protein structures are chemically and structurally related and seem to be very stable.
Since a 100-fold molar excess of HOCl was necessary to induce the antiviral activity, all the reactions and modifications documented at low HOCl-concentrations might not explain the mechanism by which our HOCl-modified serum albumins inhibit viral entry of HIV-1 as well as West Nile virus. However, our study supports the hypothesis that HOCl might be part of the host defense against pathogens and apart from its direct toxic activity it may have an additional indirect but important activity, the transformation of a protein into an antiviral weapon . From the cell culture experiments of Chase et al. and Klebanoff et al. [11, 12], in which activated PMN cells release MPO and HOCl was generated, HOCl was suggested as the HIV-1-killing agent. Based on our results it is conceivable that the generated HOCl reacts with bovine serum albumin, which is usually present in high concentrations in cell culture media, and therefore mBSA would be present during viral infection. As we have now demonstrated, mBSA has a strong antiviral activity and might be also responsible for the antiviral effects observed in PMN cell cultures. The biological relevance of HOCl-modification of serum albumin or other proteins might be questionable since virus replication in vivo is not limited to inflammatory loci, where MPO is expressed in vivo leading to the production of HOCl. To our understanding, the discovery of HOCl-modified serum albumin as an antiviral agent opens a new field for the development of non-toxic agents which can be added to blood products or can be used as a local microbicide to prevent viral transmission. Since the neutralizing activity of HOCl-modified serum albumins was documented against West Nile virus and the HIV-1 NL4-3 mutants, irrespective to their coreceptor type, future work will show how efficient HOCl-modified serum albumins can neutralize other HIV-1 and HIV-2 variants as well as other viral species.
Taken together, the generation of HOCl-modified serum albumins is very simple, cost effective and needs no highly specialized laboratory equipment. We suggest that HIV-1 neutralization by HOCl-modified serum albumins is the result of masking the gp120 molecule. We have shown that the CD4 binding site and other regions, like the V3 loop, are partially masked by HOCl-modified serum albumins. Masking the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when applied, for example, as a topical microbicide.