Neutralization is the ability of antibody to bind to and inactivate virus infectivity under defined conditions in vitro. Most neutralizing antibodies also protect animals in vivo, but protection is more complex as it also involves interaction of antibody with cells and molecules of the innate immune system. Neutralization by antibody can be mediated by a number of different mechanisms: by aggregation of virions, destabilization of the virion structure, inhibition of virion attachment to target cells, inhibition of the fusion of the virion lipid membrane with the membrane of the host cell, inhibition of the entry of the genome of non-enveloped viruses into the cell cytoplasm, inhibition of a function of the virion core through a signal transduced by an antibody, transcytosing IgA, and binding to nascent virions to block their budding or release from the cell surface. The mechanism of neutralization is determined by the properties of both a virion epitope and the antibody that reacts with it. Further, since a virus has at least several unique epitopes sited in different locations on the virion, and since the paratope and other properties of the reacting antibody can vary, this means that a virus can be neutralized by several different mechanisms. Understanding the processes of neutralization informs the creation of modern vaccines, and gives valuable insights into virus-cell interactions.

SAR1 is a new IgG2a murine monoclonal antibody derived by immunization with a plant virus expressing the sequence GERDRDR from the C-terminal tail of the gp41 transmembrane glycoprotein of human immunodeficiency virus type 1 (HIV-1). SAR1 binds to peptides and proteins carrying the GERDRDR sequence, to some but not all preparations of purified virus, and to cells infected with all viruses tested. In a standard neutralization assay, SAR1 failed to neutralize, or neutralized poorly, a number of T cell line-adapted viruses. However, it was more effective at postattachment neutralization. This was measured by two assays, the inhibition of the syncytium production by input virus, and the inhibition of the production of infectious progeny virus. In general SAR1 was more effective at neutralizing progeny virus than inoculum virus. Fifty percent inhibition of progeny virus production by different HIV-1 strains was obtained with 2-26 microg/ml of SAR1. The SAR1 neutralizing epitope was mapped specifically to the gp41 C-terminal tail. SAR1 is an unusual, if not unique, antibody whose activity supports the view that part of the gp41 C-terminal tail is exposed on the outside of the virion.

The C-terminal tail of the gp41 transmembrane glycoprotein of the human immunodeficiency virus type 1 (HIV-1) virion is usually thought to be inside the virion, but it has been shown recently that part of the tail is exposed on the virion exterior. Here, using a panel of antibodies, it was demonstrated that the same part of the tail is exposed on the surface of HIV-1-infected C8166 lymphoblastoid cells and HeLa cells infected with a gp41-expressing vaccinia virus recombinant. Both types of infected cell failed to react with p17 matrix protein-specific IgGs until permeabilized with saponin, confirming the integrity of the plasma membrane. Cell-surface exposure of the gp41 tail was independently demonstrated by inhibition of HIV-1-mediated cell–cell fusion by one of the gp41 tail-specific antibodies. These data also implicate the exposed region of the gp41 C-terminal tail either directly or indirectly in the viral fusion process. Its surface exposure suggests that the gp41 C-terminal tail may be a candidate for immune intervention or chemotherapy of infection.

The envelope protein of human immunodeficiency virus type 1 (HIV-1) comprises the outer gp 120 SU domain and the anchoring gp41 TM domain, and the conventional view is that it has a single transmembrane region with the following C-terminal sequence situated entirely within the virion. However, we have recently proposed that the gp41 C-terminal region comprises three transmembrane regions and an external loop structure. Part of this loop is the peptide 731PRGPDRPEGIEEEGGERDRDRS752 that carries three antibody epitopes, 734PDRPEG739, 740IEEE743, and 746ERDRD750. PDRPEG is not detected in virions but reacts with its cognate MAb (C8) in Western blots, IEEE is a linear and non-neutralizing epitope, and ERDRD is a conformational and neutralizing epitope. Here we show that escape mutants selected with neutralizing ERDRD-specific antibody had a single 732R-->G substitution, 14 residues upstream of the cognate epitope, and no longer bound the selecting antibody. The same amino acid substitution altered epitope PDRPEG in the virion so that it now reacted with MAb C8, but left epitope IEEE unaffected. Introduction of 732R-->G by site-specific mutagenesis into the gp41 of cloned HIV-1 NL4-3 virions allowed them to escape neutralization by ERDRD-specific IgG, and confirms that 732R makes a major contribution to the neutralizing conformation of the 731-752 region of the C-terminal tail of gp41.

Evidence has been presented which shows that part of the C-terminal tail of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a neutralization epitope and is thus exposed on the external surface of the virion. Here, SAR1, a monoclonal antibody, which was stimulated by immunization with a plant virus expressing 60 copies of the GERDRDR sequence from the exposed gp41 tail, and has an unusual pattern of neutralization activity, giving little or no neutralization of free virions, but effecting modest post-attachment neutralization (PAN) of virus bound to target cells was investigated. Here, the properties of PAN were investigated. It was found that PAN could be mediated at 4 or 20 6C, but that at 20 6C maximum PAN required virus-cell complexes to be incubated for 3 h before addition of antibody. Further PAN appeared stable at 20 6C and could be mediated for at least 5 h at this temperature. In contrast, when virus-cell complexes formed at 20 6C but then shifted to 37 6C for various times before addition of SAR1, PAN was maximal after just 10 min, and was lost after 30 min incubation. Thus, PAN at 37 6C is transient and temperature-dependent. Since this scenario recalled the temperature requirements of virus-cell fusion, fusion of HIV-1-infected and non-infected cells was investigated, and it was found that SAR1 inhibited this process by up to 75 %, in a dose-dependent manner. However, antibodies to adjacent epitopes did not inhibit fusion. These data confirm the external location of the SAR1 epitope, implicate the gp41 C-terminal tail in the HIV-1 fusion process for the first time, and suggest that SAR1 mediates PAN by inhibiting virus-mediated fusion. INTRODUCTIONThe mature envelope protein of the virion of human immunodeficiency virus type 1 (HIV-1) is a trimer of heterodimers formed of the gp120 outer subunit and the gp41 transmembrane (tm) subunit. The gp120 serves to attach virus to the CD4 primary receptor and to the CXCR4 or CCR5 co-receptors on the surface of target cells, while the gp41 enables the viral and cell lipid bilayers to fuse together, resulting in the viral genome and associated proteins entering the cytoplasm and infecting the cell. Both gp120 and gp41 carry antibody neutralization epitopes (Levy, 1998).The gp41 subunit of HIV-1 and simian immunodeficiency viruses comprises the ectodomain, the tm domain, and a long C-terminal tail of approximately 150 aa residues (Gallaher et al., 1992). There is structural information on the ectodomain (Caffrey et al., 1998;Chan et al., 1997; Malashkevitch et al., 1998;Tan et al., 1997;Weissenhorn et al., 1997), but not on the C-terminal tail. The limits of the tm domain are not known exactly (West et al., 2001). Conventionally the tail is regarded as being located entirely inside the virion or the infected cell, but it has been proposed that about 40 residues of the tail region are looped out to the external surface of the virion (Cleveland et al., 2003;McLain et al., 2001). The main evidence for this relates to the neutra...

The antibody-binding site, through which an antibody binds to its epitope, is a complex structure formed by the folding together of six complementarity-determining regions (CDRs). However, certain peptides derived from CDR sequences retain antibody specificity and function; these are know as microantibodies (MicroAbs). For example, the F58 MicroAb is a 17 residue, cyclized peptide (CDLIYYDYEEDYYFDYC) derived from CDR-H3 of F58, an IgG1 specific for the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Both MicroAb and IgG recognize the same epitope in the V3 loop and, despite its small size, the MicroAb neutralizes the infectivity of HIV-1 IIIB only 32-fold less efficiently on a molar basis. The advantage of MicroAbs is that their small size facilitates structure-function analysis. Here, the F58 MicroAb was investigated using alanine scanning, mass spectroscopy and surface plasmon resonance. Neutralization of infectious IIIB was generally more sensitive to alanine substitution than binding to soluble gp120. There appeared to be a division of function within the MicroAb, with some residues involved in antigen binding (alanine substitution of 11D, 12Y or 13Y abrogated both binding and neutralization), whereas others were concerned solely with neutralization (substitution of 3L, 8Y or 14F abrogated neutralization, but not binding). The MicroAb is predominantly b-sheet and has strong conformational constraints that are probably essential for activity. The MicroAb and soluble gp120 formed a 1 : 1 complex, with an association rate that was threefold greater than that with IgG and a faster dissociation rate. Its equilibrium dissociation constant is 37?5-fold greater than that of IgG, in line with neutralization data. This study demonstrates how MicroAbs can make a useful contribution to the understanding of antigen-antibody interactions. INTRODUCTIONAn antibody binds to its cognate epitope through its paratope, a domain formed from the six complementaritydetermining regions (CDRs) situated within the variable portions of the immunoglobulin light and heavy chains. Thus, a bivalently binding IgG can attach to antigen by the combined action of 12 CDRs. A microantibody (MicroAb) is the minimum recognition unit of an antibody and comprises a peptide derived from usually just one CDR. Its activity will depend on both its sequence and conformation. Some antiviral MicroAbs retain the virus binding and infectivity neutralizing activities of the parent antibody. The small size of MicroAbs should enable residues that are key to virus binding and neutralization activities to be easily identified. MicroAbs also have a potential advantage over IgG as therapeutic antivirals as they are less immunogenic and should be useful in situations where larger antibodies cannot easily penetrate.MicroAbs have proved difficult to identify. This may be because the potential MicroAb does not mimic the conformation of the CDR sufficiently well, because effective IgG binding requires more than one CDR or because the mass of ...

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We have read with interest the recent publication of Aygören-Pürsün et al. (1) in which they ascribe 5 seroconversions for human parvovirus B19, among 16 previously untreated and susceptible persons receiving recombinant factor VIII, to possible B19 contamination of the albumin excipient. A more likely explanation, in the absence of a close temporal association with infusion of a particular product batch, is that this just represents community-acquired infection of this endemic virus, particularly in a group of young age. We have suggested this previously (2-4) and such an explanation would also be supported by a study we performed in 1995 showing similar age dependence of B19 seroprevalence in the Scottish population and Scots persons with haemophilia (Fig. 1), and by other studies that include an appropriate control group, such as that of Williams et al. (5).