Immunogenicity of the outer domain of a HIV-1 clade C gp120
© Chen et al; licensee BioMed Central Ltd. 2007
Received: 06 April 2007
Accepted: 17 May 2007
Published: 17 May 2007
The possibility that a sub domain of a C clade HIV-1 gp120 could act as an effective immunogen was investigated. To do this, the outer domain (OD) of gp120CN54 was expressed and characterized in a construct marked by a re-introduced conformational epitope for MAb 2G12. The expressed sequence showed efficient epitope retention on the isolated ODCN54 suggesting authentic folding. To facilitate purification and subsequent immunogenicity ODCN54 was fused to the Fc domain of human IgG1. Mice were immunised with the resulting fusion proteins and also with gp120CN54-Fc and gp120 alone.
Fusion to Fc was found to stimulate antibody titre and Fc tagged ODCN54 was substantially more immunogenic than non-tagged gp120. Immunogenicity appeared the result of Fc facilitated antigen processing as immunisation with an Fc domain mutant that reduced binding to the FcR lead to a reduction in antibody titre when compared to the parental sequence. The breadth of the antibody response was assessed by serum reaction with five overlapping fragments of gp120CN54 expressed as GST fusion proteins in bacteria. A predominant anti-inner domain and anti-V3C3 response was observed following immunisation with gp120CN54-Fc and an anti-V3C3 response to the ODCN54-Fc fusion.
The outer domain of gp120CN54 is correctly folded following expression as a C terminal fusion protein. Immunogenicity is substantial when targeted to antigen presenting cells but shows V3 dominance in the polyvalent response. The gp120 outer domain has potential as a candidate vaccine component.
The need for a form of HIV-1 envelope protein capable of eliciting a broadly neutralising antibody (NAb) response as part of an HIV vaccine has been widely discussed [1–3]. It is generally agreed that the lack of NAb is a consequence of a number of evasion mechanisms evolved by the virus to maintain immunological silence. Examples include glycan shrouding  and envelope structural heterogeneity [5, 6]. Envelope structural heterogeneity involves primarily the outer envelope protein gp120. The monomeric molecule is flexible, with comparisons between the crystal structures of liganded and unliganded gp120 showing significant local change [7, 8]. Of the three defined structural domains, the inner domain, bridging sheet and the outer domain (OD), both the inner domain and bridging sheet rearrange substantially upon CD4 binding . The inner domain and bridging sheet are also the source of heterogeneity within monomeric gp120 in solution  and have sufficient flexibility to allow structural complementation between adjacent molecules [9, 10]. There is additional heterogeneity in the envelope molecules on the virion surface where gp120 and transmembrane gp41 make up the virion spike which appears patchily distributed and in a variety of conformations [5, 11, 12]. Strategies for improving gp120 immunogenicity have been reviewed recently . One approach has examined the gp120 OD in isolation as, by contrast with the complete molecule, it is structurally stable . The OD is heavily glycosylated and relatively immunologically silent in infected individuals but its potential to act as a generator of NAb is demonstrated by the fact that the epitope for a broad ranging, neutralizing human monoclonal antibody, 2G12, maps to it [15, 16] as do a number of lectins which potently neutralize virus infectivity in vitro [17, 18]. MAb 2G12 is unique in that recognition of its epitope, a high-mannose carbohydrate cluster on gp120, is achieved through a dimeric antibody structure in which there is VH exchange between adjacent immunoglobulin molecules . The paucity of this form of Ig structure in total IgG  suggests that a similar specificity may be difficult to generate following immunisation but, as the juxtaposition of key N-linked glycans is essential for MAb binding, the 2G12 epitope does provide a sensitive measure of OD conformation. Yang et al., showed that the OD (residues 252RPVVST.....DNWRS482) of gp120 from B clade virus YU2, which they termed OD1, retained 2G12 binding despite being poorly detected by the majority of HIV positive sera [14, 21]. Interest in the domain lies in the fact that 2G12 does not directly compete for the primary receptor binding site on gp120  but appears to impair secondary receptor binding . In addition, gp120-2G12 complexes exhibited reduced binding to DC-SIGN, consistent with antibody capping of at least some of the mannose moieties that would otherwise bind the lectin . Such in vitro inhibition of gp120 receptor binding is of consequence in vivo as 2G12, in combination with other neutralizing MAbs, such as b12 (against the CD4 binding site ) and 2F5 (against gp41), provides protection against HIV-1 challenge in animal models [24–26]. M-type HIV-1 includes 9 clades [27–29] and vaccine candidates designed for beneficial antibody induction should provide immunity against all clades if they are to be effective . It has been noted that C clade envelopes are significantly different to those of B clade isolates, especially early in infection [30, 31] and, more generally, that subtype C candidate vaccine development has been reportedly more problematic than clade B focused strategies . HIV-1 C clade isolates have frequently lost the carbohydrate sites required by 2G12 and so rarely present the epitope . It was unclear therefore if data obtained with the OD of the B clade gp120YU2  would be repeated using the OD of a C-clade isolate nor yet how a meaningful conformation of the latter OD could be confirmed in order to test such a possibility. Here, using the gp120 sequence of HIV-1CN54, a B/C clade recombinant originally isolated in China [34, 35], we examine the C clade gp120 OD using a re-engineered 2G12 epitope to provide a measure of conformational relevance before use as an immunogen. Subsequently, Fc tagged OD is used as an immunogen in a comparative study with gp120-Fc and untagged gp120.
Results and discussion
Characterisation of the 2G12 epitope reconstructed in clade C gp120
The 2G12 epitope is maintained on the isolated outer domain
The juxtaposition of mannose residues on the termini of key gp120 glycans creates the 2G12 epitope which is therefore conformational [15, 16, 21, 33]. Yang et al., described maintenance of the 2G12 epitope on the OD of HIV-1YU2  which also defined the OD as conformationally relevant. To assess if this was also true of the re-engineered 2G12 epitope on gp120CN54+, the OD of gp120CN54 (residues251IKPV...NWRS481) was amplified from a clone encoding gp120CN54+, cloned (additional file 1B)
Immunogenicity of ODCN54+-Fc, gp120CN54+ and gp120CN54+-Fc
This data confirms that the outer domain of gp120 in isolation can be immunogenic despite not normally generating significant responses in HIV-1 infected individuals. Similarly, the finding that the majority of the response was to polypeptide not carbohydrate despite the hyperglycosylation of the OD  is supported by the reaction of the sera generated with GST-gp120 fusion proteins expressed in bacteria. However, our data do not confirm the conclusion that the OD of HIV-1YU2, upon immunisation, resulted in few antibodies to the V3 loop . That conclusion was inferred from serum depletion experiments and no direct epitope mapping was done whereas our use of overlapping fragments of gp120CN54 as antigen suggests that, for the ODCN54+, induction of a strong response to the V3C3 region of gp120 took place. The pattern of reactivity was the same irrespective of the titre of the serum obtained suggesting that this region is a prominent feature of the CN54 sequence.
The finding that OD-Fc fusions are improved immunogens is consistent with a mechanism of enhanced immunogenicity through engagement of the FcR on antigen presenting cells. The reduction in titre following immunisation with ODCN54+-Fc(VA) supports this conclusion as does the lack of enhancement by further oligomerization through GG7 cross linking. Although reduced, the serum titre in response to ODCN54+-Fc (VA) was still far higher than ODCN54+-His alone probably as a result of a residual level of FcR binding. Within the context of IgG1 the VA mutation leads to a 10–100 fold reduction in FcR binding  effectively meaning that 1–10% of the ODCN54+-Fc(VA) immunogen was capable of generating a serum response which was observed to be ~50% of the level obtained with the standard dose of non mutated ODCN54+-Fc. This suggests that gp120-Fc fusions would be immunogenic at significantly lower doses than those used here (10 μg), an important consideration in vaccine design.
The response to gp120CN54+-Fc included reaction with all 5 fragments probed but a predominant response against the N terminus (the C1 domain) and a fragment encoding the V3 loop. Reaction with the C1 domain is consistent with the flexible gp120 inner domain which stimulates antibodies that are non-neutralising . The OD of the C clade gp120CN54+ presented a 2G12 epitope that bound MAb as well as the full length protein (or equivalent amounts of a B clade gp120 – not shown). As the spatial juxtaposition of glycans on residues 295 and 392 is crucial for MAb binding we infer from this that the OD of gp120CN54+ may be structurally very similar to that of the solved B clade structures [7, 8, 23]. Novel mannosylated compounds able to bind 2G12 have been suggested as synthetic immunogens that may be able to elicit 2G12 like antibody responses [50, 51]. A framework based on the natural target, the gp120 OD domain, would be preferable if immunogenicity could be enhanced and the Fc fusion reported here is a simple method of achieving this with the aim of generating further antibodies with 2G12 like properties. In addition, reaction with the V3C3 region was substantial suggesting that substitution of the V3 loop within ODCN54 with other conserved neutralising epitopes, such as those from gp41, may allow their prominent exposure for the generation of broadly cross clade neutralising Abs as has been attempted recently by grafting into the V1/V2 region of gp120 .
The 2G12 epitope is faithfully reconstructed in a clade C gp120 backbone with 2 glycosylation site additions. The epitope is retained on the C clade outer domain where it acts as a marker of outer domain conformation. A C-terminal Fc tag provided for enhanced immunogenicity in the absence of any other adjuvants for full length C clade gp120 and for the normally immunologically silent outer domain. The serum response to the outer domain indicated a prominent V3C3 presentation for the clade C molecule in contrast to what has been observed for the clade B outer domain. Conformationally defined gp120 fragments as immune complexes may have potential as part of a vaccine design strategy targeting humoral immunity.
Cells and manipulations
E. coli Top10 was used for the propagation of plasmids and all cloning. Expression using recombinant baculoviruses used Spodoptera frugiperda (Sf9) insect cells unless otherwise stated. Sf9 cells were cultured in SF900-II (Life Sciences) at 28°C. A description of the cloning and mutagenesis steps is shown in additional file 1.
Recombinant baculovirus infections
Infections for virus growth were done at an MOI of 0.1 and for protein expression, an MOI of 3. Virus growth was typically for 4 days or until there was considerable cytopathic effect. Sf9 cells infected for protein expression were harvested 72 hours post infection and the glycosylated protein present in the supernatant purified as described.
Microtitre plates (Thermo Labsystems) were coated with purified proteins previously normalised, blocked with PBS, 5% dried milk powder and used immediately. Primary antibodies were incubated with antigen for 60 min at room temperature. Unbound antibody was removed by washing five times with PBS containing 0.05% v/v Tween-20 and the plate was incubated with HRP-conjugated anti-mouse antibody (1:1000, Chemicon) for one hour at room temperature. The plate was washed extensively and incubated with 3,3',5,5'-Tetramethylbenzidine (TMB) chromagenic substrate (Europa Bioproducts). The reaction was stopped by addition of an equal volume of 0.5 M HCl and the absorbance was read at 410 nm.
Protein samples were separated on pre-cast 10% Tris.HCl SDS-polyacrylamide gels (BioRad) and transferred to Immobilion-P membranes (Millipore) using a semi-dry blotter. Filters were blocked for one hour at room temperature using TBS containing 0.1% v/v Tween-20 (TBS-T), 5% w/v milk powder. Primary antibody was used at a dilution of 1:500 in PBS-T, 5% w/v milk powder unless otherwise stated. Following several washes with TBS-T the membranes were incubated for 1 hour with HRP-conjugated anti-mouse antibody (Chemicon) and the bound antibodies detected by BM chemiluminescence (Roche).
We thank Herman Katinger and Dennis Burton for the original supplies of 2G12 and b12 respectively and the UK Centre for AIDS Reagents (EU Programme EVA/AVIP) for other HIV reagents. We thank Pam Rummings and Trevor Jenkinson for help with the immunisations. Funding was from the UK Medical Research Council (MRC) and the FP6 microbicide programme of the European Union (EMPRO).
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