Vaccine 20 (2002) 1169–1180

Construction of recombinant targeting immunogensincorporating an HIV-1 neutralizing epitope into sites

of differing conformational constraint

Jason Hoa, Kelly S. MacDonalda,b,c, Brian H. Barbera,∗a Department of Immunology, Medical Sciences Building, 1 Kings College Circle, University of Toronto, Toronto, Ont., Canada M5S 1A8

b Department of Microbiology, Mount Sinai Hospital, Toronto, Ont., Canadac Department of Medicine, University of Toronto, Toronto, Ont., Canada

Received 8 June 2001; received in revised form 28 September 2001; accepted 28 September 2001

Abstract

2F5 is one of the very few monoclonal antibodies with the capacity to neutralize a wide spectrum of type 1 human immunodeficiencyvirus (HIV-1) strains and primary isolates. Constructing an immunogen that contains a conformational mimic of the epitope recognizedby 2F5 could provide the means to induce a broadly neutralizing anti-HIV-1 antibody response. Thus, in an effort to create a targeted,adjuvant-independent immunogen able to induce a 2F5-like antibody response, the gp41 sequence recognized by 2F5 (ELDKWAS) wasgenetically incorporated into different regions of an antibody specific for a framework determinant on human leukocyte antigen (HLA)-DR.All constructs were expressed, secreted from Sf9 insect cells, and found to retain the anti-HLA-DR specificity of the parental antibody. Threeof the four constructs in which the ELDKWAS sequence was incorporated into a�-turn (BT)-like conformational site were recognizedby the 2F5 antibody. In contrast, none of the five constructs with the same sequence incorporated into surface-exposed regions of helicalturn had any detectable 2F5 reactivity. In addition to demonstrating the significant plasticity of several regions in the antibody moleculein terms of accepting foreign sequences without loss of expression or binding specificity, these results also suggest that the native epitoperecognized by the 2F5 antibody may be more�-turn-like than helical in conformation. Importantly, with respect to vaccine development,the 2F5-reactive antibody constructs represent candidate immunogens for the adjuvant-independent induction of an HIV-1, neutralizing2F5-like antibody response in humans. © 2002 Elsevier Science Ltd. All rights reserved.

Keywords:Antibody engineering; Immunotargeting; HIV-1; gp41

1. Introduction

One of the major problems facing the development ofa vaccine against type 1 human immunodeficiency virus(HIV-1) is the high degree of sequence variability amongdifferent HIV strains and primary isolates. To date, onlythree identified monoclonal antibodies—2F5 [1], IgG1b12[2], and 2G12 [3]—have been shown to have broad neu-tralizing capacity against primary isolates across the HIV-1clades [4]. Of these, only 2F5 has a contiguous epitope, iden-tified as amino acid sequence ELDKWAS in the ectodomainof gp41, corresponding to residues 662–668 of gp160. It wasfirst isolated as a human monoclonal IgG3 antibody in 1993from a panel of transformed peripheral blood mononuclearcells obtained from HIV-1-positive patients [1,5]. The epi-tope sequence is conserved in approximately 72% of HIV-1

∗ Corresponding author. Tel.:+1-416-667-2445; fax:+1-416-667-2453.E-mail address:brian.barber@utoronto.ca (B.H. Barber).

strains, and there are few escape mutants known [1]. Therelatively low frequency of mutant emergence points to thegenetic stability of the epitope in the face of immune se-lection, an aspect that is crucial in considering the potentialeffectiveness of any given epitope as a vaccine target.

Several groups have attempted to induce anti-ELDKWASresponses by inserting the epitope sequence into various im-munogenic molecules including Hepatitis B surface antigen[6], gp120 [7], orE. coli derived MalE protein [8]. Althoughsome of them succeeded in inducing a response that rec-ognizes the corresponding synthetic peptide, most anti-seraso generated fail to neutralize HIV-1 [4,6–8]. These resultssuggest that the conformational context in which the 2F5epitope sequence is displayed on the construct is likely tohave a profound effect on whether or not the antibody re-sponse induced by the recombinant vaccine is neutralizing.Unfortunately, the three-dimensional structure of the ac-ceptor region for most of these engineered immunogens isunknown. As well, there is little information on the structure

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.PII: S0264-410X(01)00441-8

1170 J. Ho et al. / Vaccine 20 (2002) 1169–1180

of the ELDKWAS sequence in gp41, and how it changesfrom the moment of gp120-CD4 binding to membrane fu-sion. For these reasons, it is difficult to assess the conforma-tional context required for generating an HIV-1 neutralizingELDKWAS-specific response. The present study representsan effort to mimic the conformational state of the 2F5 neu-tralizing epitope by incorporating it into different sites ina monoclonal antibody specific for a non-polymorphic de-terminant in the human leukocyte antigen (HLA)-DR geneproduct. The reason for the choice of this protein frameworkis two-fold. First, the three-dimensional structure of theantibody molecule is well understood, making it easier toselect solvent-accessible sites for incorporating the epitope.Second, based on our earlier work, it has been establishedthat antigens coupled to an anti-class II major histocom-patibility complex (MHC) antibody can induce humoralresponse in vivo without requiring an adjuvant. This immu-nization strategy is known as immunotargeting [9–13], andhas been reviewed elsewhere [14]. Of particular significanceto HIV-1 vaccine development is the observation that bothsystemic IgG and secretory IgA responses can be inducedby immunotargeted antigens delivered at mucosal surfaceswithout adjuvant [15]. Mascola et al. have demonstratedthat passively transferred 2F5, together with monoclonal an-tibody 2G12, or with 2G12 and HIV immune globulin, canprotect macaques against subsequent vaginal challenge withthe highly pathogenic SHIV89.6PD [16]. The ability to in-duce a strong 2F5-like IgA response at mucosal sites couldconstitute an important consideration in developing a vac-cine strategy for sexually transmitted diseases such as HIV.

Our comparison of 2F5 reactivity to the ELDKWASsequence incorporated in regions of different secondarystructures in an immunotargeting antibody indicates thatrecognition of this continuous sequence by 2F5 dependson the conformational context in which it is incorporated.Specifically, three of the four constructs in which the EL-DKWAS sequence was incorporated in sites that imposea putative�-turn (BT)-like conformational constraint wererecognized by the 2F5 antibody. In contrast, none of the fiveconstructs in which the same sequence was incorporatedinto helical regions in the wild type (WT) molecule couldbe recognized by the antibody. The 2F5-reactive constructs,thus, represent candidate immunogens for inducing potentHIV-1 neutralizing antibody responses in vivo.

2. Materials and methods

2.1. Antibodies

The F(ab′)2 and Fab fragments of the human monoclonalantibody 2F5 were prepared and provided by Dr. Emil Paiof the Department of Medical Biophysics at the Universityof Toronto as part of the Aventis Pasteur Canadian Univer-sities Research Program. These antibody fragments werebiotinylated using EZ-LinkTM NHS-LC–biotin (Pierce,Rockford, IL) according to the manufacturer’s protocol.

2.2. Construction of recombinant HC andLC-encoding plasmids

The monoclonal antibody 44H10 is a mouse antibodyspecific for a non-polymorphic determinant in HLA-DR[17,18]. The partially humanized version (IgG1/�) wasconstructed using recombinant DNA methods in which theDNA sequence encoding the HC constant region was re-placed with the human�1, and the LC constant region withthat of human�.

The ELDKWAS-encoding nucleotide sequence was in-corporated into the heavy chain or the light chain of partiallyhumanized 44H10 using oligonucleotide-based mutagenesismethods. The sequences of the oligos used in the procedureare listed in Table 1. In the construction of the gene encod-ing the heavy chain of H-HR1, aBstEII site was found 5′ tothe targeted site of incorporation. An oligo (034) containingthe BstEII site, followed by the epitope encoding sequence(EES) and the 21 nucleotides downstream of the targetedsite of incorporation was used as the forward primer. Asecond oligo (018) that primes the 3′ end of the heavy chainconstant region was used as the reverse primer. Incorporatedin the reverse primer is anXbaI site. A polymerase chainreaction (PCR) was carried out. The resulting amplicon wasdigested withBstEII and XbaI, and the digested fragmentwas used to replace the corresponding fragment in the DNAconstruct of wild type heavy chain. In the construction of thegene encoding the heavy chain of H-HR2 through H-HR5,and of H-BT1 through H-BT4, mutagenesis was carriedout by a two-step overlap extension PCR. In the first step,two PCR components were generated. One component wasmade with a forward primer (017) that primes the 5′ end ofthe signal sequence for the heavy chain gene and a reverseprimer that primes the 21 nucleotides upstream of the tar-geted site for substitution with the epitope (035 for H-HR2,037 for H-HR3, 039 for H-HR4, 041 for H-HR5, 053 forH-BT1, 118 for H-BT2, 120 for H-BT3, or 122 for H-BT4).A HindIII site was incorporated in the forward primer, andthe complement of EES was included in the reverse primer.The resulting PCR product therefore had the EES incorpo-rated at the 3′ end. The second component was generatedwith a forward primer (036 for H-HR2, 038 for H-HR3,040 for H-HR4, 042 for H-HR5, 054 for H-BT1, 119 forH-BT2, 121 for H-BT3, or 123 for H-BT4) that primes the21 nucleotides downstream of the targeted site of epitopeincorporation, and a reverse primer (018) that primes the3′ end of the heavy chain constant region. The EES wasincluded in the forward primer, and anXbaI site was incor-porated in the reverse primer. The resulting PCR product,therefore, had the EES incorporated at the 5′ end. These twocomponents were then joined together in an overlap exten-sion PCR, amplifying using the most 5′ and 3′ primers (017and 018, respectively). The resulting amplicon was thendigested withHindIII and XbaI, and cloned into Invitrogenvector pIB/V5-His (Life Technologies, Rockville, MD) atthese sites.

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Table 1List of primers used in the construction of the genes encoding the heavy and light chains of the various ELDKWAS-containing partially humanized44H10 antibodiesa

a Restriction sites and the encoded amino acid residues corresponding are indicated below the oligo nucleotide sequence. The residues encoded bythe complementary strand (in the case of reverse oligos) are italicized. See Section 2 for construction details.

1172 J. Ho et al. / Vaccine 20 (2002) 1169–1180

The plasmid containing the gene that encodes the lightchain of L-CT× 3 was provided by Dr. Farangis Saleh(University of Toronto). Briefly, it was constructed usingtwo complementary synthetic oligos (3KF and 3KR) that hy-bridize into a double-stranded DNA encoding the three tan-dem epitope repeats. ABamHI site was incorporated at the5′ end and anXbaI site at the 3′ end of the attachment. Thewild type light chain construct was digested withHindIIIandBamHI, and the shuttle vector pRc/CMV was digestedwith HindIII and XbaI. The three fragments were then lig-ated together to yield a cloning intermediate. The resultingplasmid was digested withHindIII andXbaI and the releasedinsert was cloned into Invitrogen vector pIZ/V5-His (LifeTechnologies). In the construction of the gene encoding thelight chain of L-CT, PCR was carried out with a forwardprimer (001) that primes the 5′ end of the light chain signalsequence and a reverse primer (101) that primes the 3′ endof the gene, using the plasmid containing the L-CT× 3gene as a template. AHindIII site was incorporated into theforward primer; the complement of the EES and anXbaIsite were incorporated into the reverse primer. The resultingamplicon was digested withHindIII and XbaI and clonedinto pIZ/V5-His vector via these sites. All constructs weresequenced to verify incorporation of the epitope sequence.

2.3. Transfection of Sf9 cells with recombinantDNA constructs

Sf9 cells (fromSpodoptera frugiperda) were kindly pro-vided by Dr. Tania Watts (University of Toronto). Theywere cultured in Grace’s insect media (Tissue Culture Me-dia Preparation facility, Faculty of Medicine, University ofToronto) supplemented with 10% fetal bovine serum (FBS;Cansera, Rexdale, Ontario, Canada), penicillin and strepto-mycin (Life Technologies), Glutamax II (Life Technologies),lactalbumin hydrolysate (Life Technologies), and yeastolate(Life Technologies). An amount of 2×106 cells were seededper 35 mm well in a six-well plate and allowed to attachfor 30 min. After washing with unsupplemented Grace’s in-sect media, they were transfected with 2�g of each plasmidwith TransIT Insecta (PanVera, Madison, WI) according tothe manufacturer’s protocol. Transfected cells were grownin Sf900II serum free insect media (Life Technologies). Su-pernatants were sampled on day 4 after transfection for se-cretion of the proteins of interest.

2.4. Capture ELISA for detection of secretedconstructs in Sf9 supernatant

ELISAs were carried out using Falcon ProBind assayplates (Becton Dickinson, Franklin Lakes, NJ). They werecoated with 5�g/ml of mouse anti-human IgG1 (Caltag,Burlingame, CA) in PBS with 0.02% (w/v) NaN3, and incu-bated at 4◦C overnight. The plates were then blocked with1% (w/v) BSA in PBS and incubated for 1 h at 37◦C. Af-ter washing three times with 0.05% (v/v) Tween-20 in PBS,

Sf9 culture supernatant was added and incubated at 37◦Cfor 1 h. After another washing, detection reagent was added.For light chain detection, alkaline phosphatase conjugatedgoat anti-human� (Caltag) at 1:1000 dilution was used.The assay was developed using the phosphatase substratep-nitrophenylphosphate (Sigma, St. Louis, MO) at 1 mg/mlin 10% (v/v) diethanolamine (pH 9.8). Absorbance at 405 nmwas measured in a Dyantech MRX ELISA plate reader. Fordetection of 2F5 binding, concentrations of constructs insupernatants were first normalized as follows. A dilutionseries of human IgG1/� (Sigma I-3889) were subjected tothe same capture ELISA described above. Concentrationsof the serially diluted standard and their corresponding ab-sorbance readings were fit by curvilinear regression usingKaleidaGraphTM version 3.08 (Synergy Software Technolo-gies, Essex Junction, VM) to the Robard equation

f (x) = m1 − m4

1 + (x/m3)m2+ m4

wherex is [antibody],m1 the maximum value,m2 the valueat point of inflection,m3 the slope at point of inflection,m4the minimum value. Concentrations of the constructs in su-pernatant were then determined by computed interpolation.Concentrations were equalized to 0.15�g/ml by diluting thesupernatants accordingly using PBS with 1% BSA. Usingthe normalized supernatants, further serial dilutions weremade, which were then tested for 2F5 reactivity. In the 2F5reactivity assay, the same capture ELISA was performed asdescribed above, except that the captured constructs weredetected using biotinylated 2F5 Fab at 1�g/ml and alkalinephosphatase conjugated avidin (ICN, Costa Mesa, CA).

2.5. Surface staining of C1R cells and flow cytometryfor detection of HLA-DR and 2F5 binding

C1R cells were cultured and passaged in RPMI mediasupplemented with 10% (v/v) FBS (Cansera), penicillin,streptomycin (Life Technologies), and Glutamax II (LifeTechnologies). For IgG detection, supernatants from Sf9cells containing the humanized 44H10 or the variousELDKWAS-modified humanized 44H10 constructs wereconcentrated approximately 10 times using AmiconTM

Centricon-10 (Millipore, Bedford, MA). The concentrationof the constructs were normalized using the same proce-dure outlined in the methods described earlier. An amountof 100�l of the concentrates or 3�g of an irrelevant anti-body of the same isotype (human IgG1/�) (Sigma I-3889)was used to stain C1R cells. For each sample, 5× 105

C1R cells were washed in 1 ml PBS with 2% FBS, thenincubated with the concentrated supernatant in the presenceof FITC-conjugated goat anti-human IgG (Caltag). Thecells were incubated for 1 h at 4◦C in the dark. They werethen washed with PBS, fixed with 2% paraformaldehydeand scanned by a FACScalibar flow cytometer (BectonDickinson). Acquired data were analyzed using CELLQuestversion 1.0 (Becton Dickinson). For competitive inhibition

J. Ho et al. / Vaccine 20 (2002) 1169–1180 1173

assay, the same procedure was used as above, except that5�g of murine (non-humanized) 44H10 was added tothe supernatants prior to staining. For 2F5 binding as-says, the same procedure as in IgG detection was used,except that instead of FITC-conjugated goat anti-humanIgG, 1�g/ml of biotinylated 2F5 F(ab′)2 and 1�g/mlR-phycoerythrin-conjugated streptavidin (PE-SA) (Molecu-lar Probe, Eugene, OR) was used.

3. Results

3.1. Engineering the 2F5 epitope sequence intovarious regions of the heavy chain or light chain of ananti-class II HLA antibody

The 44H10 is a mouse monoclonal antibody specific fora non-polymorphic determinant on HLA-DR [17,18]. It isalso known to cross-react with the class II MHC moleculesof other species including rabbits, ferrets and macaques [17],which makes it a valuable reagent for conducting immuno-targeting experiments in a number of animal models. Skeaet al. have demonstrated that an antibody response againstinfluenza hemagglutinin can be induced in rabbits and ferretswithout adjuvant by immunization with 44H10-conjugatedhemagglutinin [12]. The specificity of 44H10 was, there-fore, chosen in the construction of the immunogens for thepurpose of delivering the 2F5 epitope sequence. In order tominimize human anti-mouse antibody responses for the in-tended clinical setting and to prolong the in vivo half-life ofthe immunogen, the original murine constant regions werereplaced with the human�1/� constant regions using re-combinant DNA methods. This partially humanized 44H10molecule is denoted as WT in the present study.

To deliver the ELDKWAS epitope sequence in a confor-mationally constrained manner, a panel of constructs wasmade by genetically incorporating the EES, either by sub-stitution or by insertion, into the genes encoding the heavyor the light chain of the WT antibody. The target sites formodification in the ELDKWAS-containing constructs werechosen based on identified secondary structures in the WTmolecule (see Fig. 1). The intention was to incorporate theELDKWAS sequence in different molecular contexts thatare conducive to either helix or�-turn formation. Hence, theH-HR series of antibodies was designed to have sequencesin five helical regions (HR) of the heavy chain individu-ally replaced with the ELDKWAS sequence. In contrast,the H-BT series was designed to constrain the epitope ina �-turn-like conformation, by inserting the ELDKWASsequence at sites flanked by�-strands. As a comparisonwith these conformationally constrained modifications, twoadditional constructs were designed that incorporated theELDKWAS sequence with no imposed conformationalrestriction. These constructs, L-CT and L-CT× 3, had,respectively, one or three copies of the epitope attached tothe C-terminal the light chain.

Mutagenesis to introduce the ELDKWAS-encoding nu-cleotide sequence into the heavy chain or the light chainencoding gene of partially humanized 44H10 was carriedout by PCR methods. The gene encoding the heavy chain ofH-HR1 was constructed using a uniqueBstEII site upstreamof the targeted site of incorporation. Both theBstEII site andthe EES were built into the forward oligo, while a uniqueXbaI site was built into the reverse oligo that primes the 3′end of the gene encoding heavy chain. The resulting PCRamplicon was digested withBstEII and XbaI, and used toreplace the corresponding fragment in the gene for WT. Forthe genes encoding the heavy chain of the remainder of theH-HR series and all of the H-BT series, no unique cuttingsites were available near the sites of epitope incorporation.Mutagenesis was, therefore, carried out by a two-step over-lap extension PCR. The construction details of these genesare described in Section 2.

The gene encoding the light chain of L-CT× 3 wasconstructed by two complementary synthetic oligos, whichhybridize into a double-stranded DNA encoding the threetandem epitope repeats. By incorporating a uniqueBamHIsite at the 5′ end and a uniqueXbaI site at the 3′ end ofthe attachment, the double stranded fragment was ligatedto the WT light chain. The resultant fragment was then lig-ated into pIB vector viaHindIII and XbaI sites. Using thisplasmid as a template, the gene encoding the light chainof L-CT was constructed by PCR methods. All constructswere sequenced to verify incorporation of the epitope se-quence. The resulting changes in amino acid sequence areschematically represented in Fig. 2.

3.2. Expression of ELDKWAS modifiedantibody constructs

The various heavy chain and light chain constructs werecloned into the insect expression vectors pIB and pIZ, re-spectively, and constitutively expressed under the control ofthe baculovirus-derived OpIE2 promoter in transfected Sf9insect cells (Fig. 2). Supernatants were harvested 3 daysafter transfection and assayed for secretion of the recom-binant antibody constructs. As seen in Fig. 3a–c, all con-structs were secreted from transfected Sf9 cells. Moreover,the heavy chain was in association with the light chain inall constructs, as they were captured by an anti-heavy chainantibody and detected with an anti-light chain antibody. Theassociation of the two chains strongly suggests that thesemolecules were not denatured materials, but were properlyfolded immunoglobulins.

3.3. ELDKWAS sequence has differential reactivitywith 2F5 antibody when incorporated into differentregions of the antibody

In order to assess the reactivity of the various ELDKWAS-containing constructs with the 2F5 antibody, the

1174 J. Ho et al. / Vaccine 20 (2002) 1169–1180

Fig. 1. Schematic representation of the sites for incorporating the ELDKWAS sequence in the three-dimensional context of a representative antibodystructure. Enclosed on the left are the structures of (a) the Fab of a representative antibody (protein data bank (PDB) ID 1CLY), and (b) the Fc regionof a representative human IgG1 (PDB ID 1FC1). In (a), the representative Fab molecule is a chimeric Fab fragment with a mouse V region linked toa human�1/� constant region. The heavy chain is indicated in red and the light chain in grey. In (b), the two heavy chains are indicated in red andcyan, respectively. The sites of epitope incorporation are shown on the right (c–e). The sites for the H-HR constructs are indicated in blue; the ones forthe H-BT and the L-CT constructs are indicated by black dashed lines. For H-HR1, H-BT1, L-CT and L-CT× 3, the epitope sequence is incorporatedin the Fab region (c), whereas for H-HR2 through 5 and H-BT2 through 4, it is incorporated in the Fc region (d and e). For clarity, only one of thetwo heavy chains in the Fc fragment is shown in (d) and (e), which illustrate two spatial orientations of the chain. The numbers indicate the positionof the targeted amino acid residues. Numbering follows the system specified by Kabat et al. [43]. The amino acid numbers originally published for thestructure in panel (b) did not follow that system, and are hereby converted accordingly for the purpose of uniformity. For H-HR4, the seven continuousresidues in the target site are numbered 375, 376, 377, 378, 381, 382, and 383 in this system.

J. Ho et al. / Vaccine 20 (2002) 1169–1180 1175

Fig. 2. Schematic representation of the sites of incorporating the 2F5 epitope in the partially humanized 44H10 antibody. The wild type (WT) moleculewas constructed by joining the VH and VL of 44H10 with human�1 and� constant region. H-HR1 through 5 were constructed by replacing the indicatedamino acid residues which form helical turns in the WT molecule with the 2F5 epitope sequence ELDKWAS (represented by the thick line). H-BT1through 4 were constructed by inserting the ELDKWAS sequence at the various sites indicated that are flanked by�-strands, likely constraining theinserted sequence into a�-turn-like conformation. L-CT and L-CT× 3 were constructed by attaching one or three ELDKWAS sequence, respectively, tothe carboxyl terminal of the light chain. The numbers indicate the position of the residues targeted for epitope incorporation. Amino acids are numberedaccording to Kabat et al. [43]. For HR4, the seven continuous residues in the target site are numbered 375, 376, 377, 378, 381, 382, and 383 in this system.

concentration of recombinant constructs was first deter-mined and normalized against a standard curve generatedfrom a serial dilution of an antibody of known concentration(data not shown). An ELISA was then performed on thenormalized supernatants using the same procedure as in theassay for secreted constructs, except that the detection wasperformed with biotinylated 2F5 Fab fragment and alkalinephosphatase-conjugated avidin. As shown in Fig. 3d, noneof the five H-HR constructs had any detectable 2F5 reac-tivity. In contrast, three of the four constructs in the H-BTseries, as well as one of the constructs with C-terminally at-tached epitopes (L-CT× 3) showed positive binding to 2F5(Fig. 3e and f). The differential binding observed among theconformationally constrained epitope-containing constructssuggests that recognition of an ELDKWAS sequence by the

2F5 antibody is facilitated by a�-turn constraint, but notby a helical conformation.

3.4. All ELDKWAS-containing mutants retainHLA-DR binding specificity

In order for immunotargeting to be possible, the im-munogen molecule must also possess anti-class II MHCreactivity. Thus, it was important to determine whether theinsertion or substitution made in each of the mutants hadaffected the ability of the constructs to bind HLA-DR. Tothis end, supernatants of transfected Sf9 cells were used tostain C1R cells. The expression of HLA-DR on C1R cellswas confirmed by the positive staining observed with theoriginal non-humanized 44H10 (data not shown). When the

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concentrated supernatants of transfected Sf9 cells were usedto stain C1R cells, the WT as well as the ELDKWAS-modifiedantibody constructs were all found to bind to the surfaceof the cells (Fig. 4a, solid histogram). Moreover, this bind-ing could be competitively inhibited with non-humanizedmurine 44H10 (Fig. 4a, open histogram). Taken together,these data show that the modifications to the different an-tibody constructs have not ablated the parental HLA-DR

Fig. 4. All constructs retained HLA-DR specificity; H-BT2, 3 and L-CT× 3 could furthermore be recognized by 2F5 while bound to HLA-DR. (a) Indetecting the binding of the constructs to HLA-DR, supernatants containing the various constructs and FITC-conjugate goat anti-human IgG was usedto stain C1R cells (solid histograms). The specificity of staining by various constructs was shown by competitive inhibition using HLA-DR-specificnon-humanized mouse 44H10 (open histograms). (b) In assaying for 2F5 reactivity of cell-bound molecules, the same procedure was used as in (a)except that biotinylated 2F5 F(ab′)2 and streptavidin-conjugated PE (PE-SA) were used as secondary reagents to stain C1R cells. The numbers in thehistograms indicate the geometric mean fluorescence of the samples.

specificity. They also confirm that the constructs correspondto properly folded, fully functional antibodies.

Using a similar staining procedure, the supernatants werealso tested to see if the constructs that bind to the surface ofthe cells via HLA-DR can be simultaneously recognized bythe 2F5 antibody. This system would detect 2F5 reactivityonly among those molecules that are bound to the surfaceof the cells. By virtue of their binding specificity, these

1178 J. Ho et al. / Vaccine 20 (2002) 1169–1180

molecules should be functionally intact and properly folded.It rules out the possibility that any observed 2F5 reactiv-ity might have arisen from a sub-population of denaturedmolecules in the supernatants. As such, a positive signalwould be indicative of the 2F5 antibody binding to a confor-mationally constrained ELDKWAS sequence. Towards thisend, biotinylated 2F5 F(ab′)2 in conjunction with PE-SAwas used as detection reagent in staining construct-coatedC1R cells. As seen in Fig. 4b, the H-BT2, 3, and L-CT× 3molecules bound on the surface of C1R cells were found tobe recognized by the 2F5 antibody (Fig. 4b). The positive2F5 staining seen with these constructs correlates with theirpositive 2F5 reactivity seen in the ELISA system. Lowerbut detectable staining was also seen with the other uncon-strained control L-CT in this assay system. This suggests thatthe ELDKWAS sequence in this construct can also mediatelimited binding to 2F5. Based on these observations, we con-clude that the constructs H-BT2, H-BT3 and L-CT× 3 canbind to HLA-DR and be recognized by 2F5 at the same time,and that the positive 2F5 reactivity observed in these con-structs was due to functional and properly folded molecules.

Interestingly, H-BT1, the fourth 2F5-reactive constructidentified in the ELISA system, was not recognized by 2F5while it was bound to the surface of the cell. Since the EL-DKWAS sequence in this construct is inserted in the V re-gion, it is likely that the binding site for HLA-DR and theinserted epitope sequence are in too close proximity to allowsimultaneous binding. Thus, the absence of 2F5 staining inthis sample was likely a reflection of the steric hindrancebetween these two interactions. However, from the point ofview of immunotargeting immunization, binding to a pro-fessional antigen presenting cell (for subsequent process-ing and presentation) and binding to the epitope-recognizingB cell are likely independent events in vivo. Simultaneousbinding to HLA-DR and the antigen-specific B cell there-fore may not be required. Taken together, the results henceshow that H-BT1, 2, and 3 possess the necessary character-istics to qualify as candidate immunogens to deliver a con-formationally constrained 2F5 epitope by immunotargeting.L-CT × 3, on the other hand, can be considered as an addi-tional immunogen to deliver the same epitope without con-formational constraint.

4. Discussion

Various groups have demonstrated that the binding of theHIV-1 envelope protein gp120 to CD4 induces a conforma-tional change, which reveals conserved cryptic epitopes cru-cial to chemokine co-receptor binding [19–24]. Subsequentco-receptor binding causes further conformational changesin gp120 [22,23], unshielding gp41, and ultimately result-ing in the insertion of the gp41 fusion peptide into the hostcell membrane. gp41 then undergoes a fast conformationalchange into a folding intermediate, followed by a slowerstep in which two heptad repeats (amino acids 546–481 and

628–661, denoted hereby as the N- and C-terminal heptadrepeats, respectively) collapse into a six-helix bundle thatis characteristic of the putative fusion-active form ([25],reviewed in [26]). At the moment, the dynamic changesoccurring in this sequence during CD4 binding, co-receptorbinding, and membrane fusion are not well understood. Nev-ertheless, it is known that the ELDKWAS sequence, whichis the target for the pan-neutralizing 2F5 antibody, is foundin close proximity to the gp41 locus of these importantconformational changes. If the key conformational state(s)of this epitope could be faithfully captured in a protein im-munogen, it would establish the basis for a vaccine capableof inducing broadly neutralizing anti-HIV-1 antibodies.

Predictions of the secondary structure of ELDKWAS havebeen made based on primary sequence [27]. The data sug-gest that this sequence has a high helical propensity andmay exist as an extension of the C-terminal helical heptadrepeat by virtue of the absence of helix-breaking residue. Itis conceivable that at some point during the transition, es-pecially in the initial stages, that the epitope may assumea helical conformation. However, as the heptad repeats col-lapse onto themselves like three V-shaped arms and foldinto a six-helix bundle, the regions connecting the repeatsand the transmembrane anchor likely has to sterically bendinto a more loop-like structure to accommodate the perpen-dicularly juxtaposed helices of the heptad repeats and thetransmembrane domain. It is, therefore, also conceivable thatthe ELDKWAS sequence, being part of this connecting re-gion, may undergo conformational changes towards a moreloop-like structure in the collapse of the heptad repeats andthe formation of the six-helix bundle.

Thus, one of our principal objectives is to determinewhether the native 2F5-reactive ELDKWAS epitope is moreaccurately reflected by a helical or a�-turn-like confor-mation. By incorporating the ELDKWAS sequence intoregions of the antibody molecule that have helical turns,or sites that will likely constrain it in a�-turn-like mannerby virtue of flanking � strands, the series of constructsengineered is intended to provide different conformationalconstraints conducive to formation of either helical or�-turn-like secondary structures. The data show that threeof the four constructs in which the epitope is inserted into a�-turn-like site (H-BT) are reactive with 2F5, whereas noneof the constructs containing the same sequence in regionsof helical turns are 2F5 reactive. This clearly suggests thatthe native 2F5 epitope may be more�-turn-like than helicalin conformation. Interestingly, this correlates well with thecrystallography data from Pai et al., which show that theELDKWAS peptide adopts a hairpin turn when bound to theantibody [28]. It also correlates with the high 2F5 reactivityof two recombinant proteins that contain the ELDKWAS se-quence constrained in a similar conformation [29,30]. Onesuch construct was investigated earlier in our laboratory,in which the ELDKWAS sequence was inserted into the Vregion of another antibody scaffold at a site homologousto that in H-BT1. In that system, the resulting immunogen

J. Ho et al. / Vaccine 20 (2002) 1169–1180 1179

was tested in guinea pigs and shown to induce an antibodyresponse that recognized intact HIV-1 gp160 [30]. Collec-tively, these data suggest that the native epitope of the 2F5antibody exists, at least in part, as a�-turn-like conforma-tion. In order to further characterize 2F5 binding to theseantigenic structures and to investigate the structure of theincorporated sequence, efforts are currently under way tomeasure the affinity of these interactions and to determinethe three-dimensional conformation of the constructs byX-ray crystallography.

Apart from the structural issues raised by these engi-neered antibody constructs, the current study also representsan important extension of the immunotargeting vaccine de-sign strategy. Various groups have explored the delivery ofT and B epitopes in vivo by genetically incorporating theminto the framework of the immunoglobulin molecule. Thevariable region, in particular the complementarity deter-mining regions (CDRs), have provided attractive sites forgenetic incorporation of both T [31–34] and B [30,35–37]epitopes. The naturally occurring variation in both lengthand sequence in this region among antibody molecules isthought to be an indication of tolerance for inserted for-eign sequences without affecting the expression or overallfolding of the whole molecule. Such strategies have beenreviewed elsewhere [38]. In contrast, incorporation of for-eign epitopes in the constant region is usually achieved notthrough genetic recombination but through coupling meth-ods to, for instance, the sugar moieties [39,40]. A notableexception is the construct developed by Lunde et al., inwhich foreign epitopes have been genetically incorporatedin the CH1 domain of the constant region [41,42]. The datapresented in this report further support the feasibility of thelatter strategy and show that foreign B-cell epitopes canbe genetically incorporated into numerous sites in both thevariable and the constant regions of an antibody moleculewhile permitting appropriate folding and binding speci-ficity. This could serve as a model for the presentationof epitopes with a variety of conformational requirementswhile retaining adjuvant-independent immunotargetingpotential.

It is interesting that the insertion of the ELDKWAS se-quence into site 74 of the heavy chain V region wouldnot allow simultaneous binding to HLA-DR and 2F5. Ourworking model of immunotargeting predicts that the capac-ity for such simultaneous binding to class II MHC moleculeand the epitope-specific B cell would not be required. Theavailability of the H-BT1 construct now offers the oppor-tunity for a formal examination of that hypothesis. Thus,H-BT1, along with H-BT2 and 3, provide three candi-date adjuvant-independent immunogens that contain theELDKWAS sequence in a conformationally constrained,2F5-reactive manner. Despite the existence of three broadlyneutralizing monoclonal antibodies, IgG1b12, 2G12, and2F5, there has been no report of successful induction ofsuch neutralizing response in vivo by means of vaccinationwith their epitopes. We remain hopeful that the immuno-

gens identified in this paper will be able to induce ananti-ELDKWAS response in vivo, and, more importantly,that the antibodies induced will be neutralizing for a broadrange of primary HIV-1 isolates.

Acknowledgements

This work was funded by the Aventis Pasteur CanadianUniversities Research Program (CURP), the Medical Re-search Council (MRC), and a grant from the Ontario HIVTreatment Network (OHTN).

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