University of Aberdeen, Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK1
Department of Microbiology, University of Iowa College of Medicine, Iowa City, IA 52242, USA2
Scotgen Biopharmaceuticals Inc., Kettock Lodge, Aberdeen Science and Technology Park, Aberdeen AB22 8GU, UK3
Author for correspondence: Andy Porter. Fax +44 1224 273144. e-mail a.porter{at}abdn.ac.uk
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The VZV specific antibody with the highest documented neutralization titre is directed against VZV glycoprotein gH (Grose, 1990 ). Of particular interest, one of these anti-gH murine monoclonal antibodies (MAb 206) (Montalvo & Grose, 1986
) is capable of neutralizing VZV in vitro in the absence of human complement. Furthermore, blockage of viral egress and inhibition of cell-to-cell spread of VZV in cultured cells have also been demonstrated (Rodriguez et al., 1993
). Because of the large body of knowledge about this anti-gH monoclonal antibody and its apparent biological relevance, we selected it as a candidate for humanization. Moreover, we investigated whether recombinant antibody fragments could be generated that retain these neutralizing properties.
Transfer of the 206 murine CDRs to human frameworks was achieved by oligonucleotide site-directed mutagenesis (Tempest et al., 1991 ; Nakamaye & Eckstein, 1986
) to form the hu VH and hu V
sequences. The templates for mutagenesis were M13HuVHLYS, which contains VH framework regions from human antibody NEWM and VH CDRs from murine antibody D1.3 cloned into M13VHPCR1, and M13HuVKLYS, which contains V
framework regions from human antibody REI and V
CDRs from murine antibody D1.3 cloned into M13VKPCR1 (Riechmann et al., 1988
). The oligonucleotides used were: VHCDR1, 5' dTGTCT CACCC AGCTC ATCCA GAATC TACTG AAGTC GAATC CAGAC GCTGT GCAGG TCAGG 3'; VHCDR2, 5' dTTGCT GGTGT CTCTG AGCAT TGTCA CTCTG TCCTT CAGAG ATGGC ATATA GTTTA TCGTA CTGCT ATCTG GATTA ATTTC TCCAA TCCACTCA 3'; VHCDR3, 5' dCCTTG GCCCC AGTAG TCCAT AGCAT AGGAC CACCC TCTTG CACAATAA 3'; VKCDR1, 5' dTACCA GTGCA TATAA CTAGA GCCAG ATGTA CTGAC ACTTT TGCTG GCCCT ACAGG TGATGGT 3'; VKCDR2, 5' dCACAC CAGAT TCTAG GTTGG ATGCA AGGTAG 3'; VKCDR3, 5' dGGTCC CTTGG CCGAA CGTGA ATGGA AGCTC CCTAC TGTGC TGGCA GTAGTAG 3'. Use of these oligonucleotides led to a number of additional murine residues being deliberately substituted into the human variable region frameworks. Specifically, the VHCDR1 oligonucleotide incorporated framework alterations at residues 24 (Val changed to Ala: V24A), 27 (Ser to Phe: S27F) and 28 (Thr to Asp: T28D) and the VHCDR2 oligonucleotide incorporated a framework alteration at residue 71 (Val changed to Arg: V71R).
Mammalian expression vectors pSVgpt and pSVhyg (Mulligan & Berg, 1981 ), containing the hu206 VH and V
genes, respectively, together with the appropriate constant regions and control elements, were used to transfect NS0 cells (European Collection of Animal Cell Cultures, Porton, UK, ECACC no. 85110505). Humanized antibody was purified by protein A chromatography.
The biological property of greatest interest is the ability of the anti-gH antibody to neutralize infectious virus in a plaque-reduction assay (Grose et al., 1979 ). Therefore, we sought to determine whether this property was retained in the humanized antibody. An aliquot of antibody ranging from 1·2 to 50 µg in a 1 ml volume was added to 1 ml of virus suspension and the 2 ml mixture was incubated for 1 h prior to inoculation onto a 35 mm MeWo cell monolayer. When the monolayers were examined 3 days later, no more than ten small plaques were visualized in any of the five monolayers inoculated with both virus and humanized antibody in a final concentration of at least 2·5 µg/ml. The virus control exhibited more than 100 plaques. This observation was easily reproducible and suggested that the humanization procedure had been successful because the recombinant antibody retained the same complement independent neutralization property of the murine antibody.
The laboratory strain VZV-32 (Grose & Brunell, 1978 ) is a low passage virus (fewer than 20 passages) isolated from a child with chickenpox. To be assured that there was no variation in susceptibility to neutralization between wild-type VZV strains, isolates from ten other children with chickenpox, collected in the USA between the years 19771990, were tested by plaque reduction. Among these ten wild-type isolates, there were no major differences in susceptibility to neutralization compared to strain VZV-32. In general, most VZV isolates were neutralized by 1·22·5 µg/ml of humanized antibody 206.
In order to evaluate whether the humanized MAb was superior to VZIG, we purchased two vials of VZIG (Massachusetts Public Health Biologic Laboratories, Boston, MA, USA). Each vial was selected from a different lot (MVZIG-44 and MVZIG-46). The VZV neutralization assays were repeated as previously described. The total protein content of VZIG was 165 mg/ml; the initial plaque-reduction assays were carried out with the same concentrations of antibody protein, i.e. between 1 and 100 µg/ml. No inhibition of plaque formation was observed. Therefore, the amount of antibody was increased by directly adding VZIG at much larger volumes. Only when a final VZIG concentration of 6 mg/ml was attained was there a greater than 80% reduction in plaque formation. If a level of 2·5 µg/ml (167 nM) of humanized Ab is considered necessary for plaque inhibition, then a comparable level for VZIG is 6000 µg/ml. Thus, the biological activity of the humanized antibody is approximately 2400 times that of the standard VZIG preparation on a mg per mg basis. There is an obvious caveat to this comparison: even though human VZIG is prepared from whole IgG with a high anti-VZV titre, a major portion of the IgG will have specificities other than the VZV gH antigen.
To further investigate the nature of the hu206 antibodyantigen interactions and provide additional insight into the mechanism of neutralization, we cloned and expressed single-chain antibody fragments (hu206 scAb). These are similar to scFv (Bird et al., 1988 ), but in addition to VH and V
they also contain the whole human light chain constant region (hu C
), and have a molecular mass of approximately 40 kDa (Fig. 1
) (McGregor et al., 1994
). A pUC19-based periplasmic expression vector, pPM1-His (Molloy et al., 1995
), was used for expression in Escherichia coli XL-1 Blue (Stratagene).
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It appears that single-chain antibody fragments expressed in E. coli can also neutralize VZV, but only when expressed as so-called diabodies or triabodies (Holliger et al., 1993 ; Kortt et al., 1997
; Lawrence et al., 1998
), or if multimerization is induced by a secondary antibody. Similar results were obtained for Fab fragments produced by papain digestion of whole hu206 MAb purified from mammalian cells. These observations raise questions about the mechanism of neutralization of the virus by antibody at this epitope. The apparent requirement for multimerization for neutralization may be explained in various ways. Firstly, it may simply be an issue of size i.e. smaller molecules are less able to block the fusogenic function of gH, due to reduced steric hindrance. The larger MAb, and antibody fragments bound by secondary antibody, may more efficiently neutralize the virus because increased steric hindrance blocks a critical step in the fusion process. Alternatively, they may cause aggregation or cross-linking of virus envelope structures. It is, however, unlikely that the neutralization effect of 206 antibody and fragments is dependent on aggregation of the virions, because, despite its name, cell-free VZV prepared by sonic disruption of infected cells tends to be a heterogeneous mixture of virions associated with fragments of cell membrane (Cohen & Straus, 1996
). The literature also suggests that virus aggregation, although possible, is not always the most likely cause of neutralization. Mason et al. (1996)
described a single-chain antibody against foot-and-mouth disease virus (FMDV) that is only weakly neutralizing in vitro unless incubated with a secondary antibody. Aggregation of the FMDV did not appear to be the mechanism for neutralizing. Thullier et al. (1999)
report that a recombinant Fab against dengue virus neutralizes in vitro, but to a degree substantially less than would be expected given its affinity compared to the parental antibody. Like antibody 206, this antibody also binds to an envelope protein, and the authors postulate that the reduced neutralization titre was due to a decrease in steric hindrance caused by the Fab, compared to the MAb. Similarly, Lamarre & Talbot (1995)
found that while proteolytically generated Fab fragments, used at sufficiently high concentrations, were able to neutralize coronavirus infectivity both in vitro and in vivo, neutralization was significantly greater in bivalent F(ab')2 fragments, but this effect was not mediated by aggregation of virions.
The possibility that simply an increased avidity of multimeric antibody (fragments) compared to monomeric fragments is responsible for their increased neutralizing potency cannot be ruled out. Avidity refers to the apparent increase in affinity observed when multiple antigen binding sites for a given epitope are located on a single molecule. The physical basis for this effect is that when the first binding site is bound to a surface, the concentration of additional antigen binding site(s) is dramatically increased. Although any avidity effect due to multimerization forced by shortening the linker failed to manifest in the cell monolayer ELISA, it may be that the distribution or orientation of the 206 epitope is different on cell-free VZV, such that multivalent binding would become possible.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 January 2001;
accepted 30 April 2001.