Reintroduction of the 2G12 epitope in an HIV-1 clade C gp120
Chen, Hongying; Xu, Xiaodong; Bishop, Alexandra; Jones, Ian M
School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK.
Received 23 January, 2005
Revised 31 January, 2005
Accepted 18 February, 2005
Many clade C isolates of HIV-1 do not react with monoclonal antibody (MAb) 2G12, a broad-ranging human neutralizing MAb that recognizes high mannose carbohydrate groups attached to glycoprotein gp120. We reintroduced a partial and complete 2G12 epitope into a clade C background, HIV-1CN54, and examined the antibody reactivity of the resulting recombinant molecules. Two glycosylation sites recovered 2G12 binding completely, but some binding was evident after the reintroduction of a single glycosylation site at Asn295.
The antibody 2G12 is a unique human monoclonal antibody (MAb) isolated 10 years ago [1,2] that recognizes high mannose carbohydrates attached to gp120 [2–4]. Neutralization by 2G12 occurs by an uncertain mechanism as it does not compete directly for either primary or secondary receptor binding sites . MAb 2G12 is broadly neutralizing, and in combination with other neutralizing MAb, such as b12 and 2F5, can provide an effective immunity to HIV-1 challenge in animal models [6–8]. The epitope recognized by 2G12 is thus of interest to the field of vaccine design. HIV-1clade C isolates, such as HIV-1CN54 [9,10], react poorly with 2G12 as sequence variation in the epitope is apparent in up to 80% of naturally occurring isolates . Sanders et al.  reported 2G12 recognized mannose or hybrid glycans at asparagines 295, 332 and 392, whereas Scanlan et al.  favoured only the carbohydrates attached to 295 and 332. However, the crystal structure of 2G12 complexed with oligomannose suggested that the N-linked glycan attached to 295 is too distant to react with 2G12, and that contacts are more probably with glycans attached to Asn 332, 339 and 392 . Moreover, although highly specific for HIV-1 gp120, 2G12 can also bind mannose clusters synthesized on simple frameworks with no underlying polypeptide chain . Some uncertainty thus remains about the precise epitope recognized by 2G12. We have replaced the residues required to restore 2G12 binding within the CN54 background and report here the outcome for 2G12 and b12 binding.
The HIV-1CN54 env gene was sourced by us for expression of a C-clade gp120 as part of the European microbicide programme (www.empro.org.uk). Gp120 was expressed using baculovirus expression system , purified by lectin affinity chromatography , and shown to bind with high affinity to CD4 cells, to react with Env sera and was immunogenic upon immunization. Gp120CN54 reacted with b12 [14,15], but not with 2G12, consistent with residue changes at Asn 295 and Thr 394, both glycosylation sites necessary for 2G12 recognition. We used site-directed mutagenesis to replace each glycosylation site singly (V295N or A394T) and in combination (V295N + A394T). The mutated gp120CN54 coding region was expressed on the surface of infected insect cells [16,17] and also as a secreted protein. Infected cells and supernatants were confirmed for the expression of gp120CN54 and mutants thereof at 2 days post-infection by sodium dodecylsulphate–polyacrylamide gel electrophoresis and Western blot using the Env polyvalent antibody ADP423. The membrane anchored form of gp120 showed high-level expression of a molecule of approximately 110 000 Mr molecular weight in the cell fractions, whereas the same gp120 variants without the membrane anchor showed expression of a smaller antigen of approximately 90 000 Mr in both cells and supernatant. The restoration of glycosylation triplets led to the expression of marginally larger proteins consistent with carbohydrate addition to Asn 295 or 392 (mutation A394T). The degree of recovery of 2G12 binding associated with the mutations made was assayed by flow cytometry and enzyme-linked immunosorbent assay, and to probe overall conformation, b12 or soluble CD4 cells. By fluorescence-activated cell sorter, 2G12 bound significantly to V295N (mean fluorescence values of 123 compared with 48 for the wild type), detectably to A394T (mean ∼103) and most strongly to V295N + A394T (mean ∼163) (Fig. 1). Parental and gp120 variants on the insect cell surface also bound b12, but recovery of the glycan at 295 had a marginally reduced binding from a mean fluorescence of approximately 403 for the parental molecule to approximately 315 for the single mutant and approximately 239 for the double mutant. Mutation at 394 reduced b12 binding less (mean fluorescence ∼339). The trend observed in b12 binding was maintained when cells were incubated with soluble CD4 cells, although the loss of binding associated with V295N (singly or in combination A394T) was less than that shown with b12, typically approximately 10% (not shown). The relative order of binding was also apparent using purified soluble antigen (Fig. 1). The double mutant V295N + A394T bound 2G12 strongly, whereas each restored single glycosylation site showed a small increase in 2G12 binding at high MAb concentrations. Mutation V295N restored a greater degree of binding than mutation A394T (Fig. 1). Similarly, wild-type gp120CN54 bound b12 marginally better than the single site mutation V295N. In both assays, the restoration of two sites gave complete 2G12 binding recovery, whereas one carbohydrate site restored binding somewhat (order V295N + A394T >>>>> V295N > A394T). However, 2G12 epitope recovery was associated with a slightly reduced ability to bind the conformational monoclonal antibody b12.
The precise epitope recognized by 2G12 is important if engineered immunogens able to elicit 2G12-like antibodies upon immunization are to be developed. Earlier studies identified an epitope consisting partly or wholly of carbohydrate [2–4,18], and the 2G12 crystal structure revealed a novel structure in which two antibodies combine to make an active binding site through a VH domain exchange . However, whereas the earlier biochemical studies indicated that Asn 295 was the favoured direct contact, Calarese et al.  concluded that it was too distant to be involved. Instead, direct contacts were proposed with N-linked glycans at 332 and 392, the glycan at 295 acting to occlude glycan 332, to prevent further trimming by glycotransferases and so stop the assembly of a complex glycan structure. In our experiments, more recovery of 2G12 binding was associated with 295 rather than 394. Moreover, insect-derived glycans are uniformly high mannose , so the requirement for the glycan at 295 to act as a shroud against the synthesis of a complex glycan at 332 does not arise. Together, our data suggest that the glycan attached to 295 is the more likely direct contact, although how this can be accommodated sterically with a distant glycan at 392 remains to be determined. In addition, the reintroduction of the 2G12 epitope to gp120CN54 was associated with reduced reactivity at the conformationally sensitive CD4 cell binding site. It is possible therefore that the apparently selective loss of 2G12 binding in many field C clade isolates  is associated with marginal increases in receptor binding and clade success. Novel antigens able to bind 2G12 with high affinity have been described [11,20]. However, as 2G12 is not present in a large pool of human IgG , the occurrence of dimeric antibodies with a VH domain exchange, rather than the immunogen, may represent the greater limitation. In which case, further analysis of the precise structure of the epitope and the method of neutralization is warranted.
The authors would like to thank Herman Katinger and Dennis Burton for the original supply of 2G12 and b12, respectively, and the UK AIDS reagent repository (www.nibsc.co.uk) for the supply of general HIV reagents.
Sponsorship: The work was funded by the UK Medical Research Council and EMPRO, a FP6 programme of the European Union.
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