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The Journal of Immunology, 2002, 169: 595-605.
Copyright © 2002 by The American Association of Immunologists

Efficient Isolation of Novel Human Monoclonal Antibodies with Neutralizing Activity Against HIV-1 from Transgenic Mice Expressing Human Ig Loci1

Yuxian He*, William J. Honnen*, Chavdar P. Krachmarov*, Michael Burkhart*, Samuel C. Kayman*, Jose Corvalan{dagger} and Abraham Pinter2,*

* Laboratory of Retroviral Biology, Public Health Research Institute, Newark, NJ 07103; and {dagger} Abgenix, Inc., Fremont, CA 94555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIVSF162 resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIVSF162 strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of a protective vaccine against HIV-1 has been hindered in part by limited knowledge of the target sites on the HIV envelope proteins, gp120 and gp41, that mediate potent neutralization of primary strains of the virus. Evidence for the existence of such targets is provided by the observation that sera of some infected people contain Abs that strongly neutralize primary isolates (1, 2, 3, 4); however, current vaccine candidates have not been able to induce similar activities (5, 6, 7, 8, 9, 10, 11). One approach for identifying the targets to which the Abs responsible for the robust neutralization seen with patient sera is to isolate mAbs that neutralize primary isolates and then map their epitopes. Despite considerable effort, however, relatively few human mAbs (hu-mAbs)3 have been described with such activities (12, 13, 14, 15).

Most available hu-mAbs were derived by EBV transformation of B cells obtained from HIV-1-infected patients and/or fusion with appropriate myeloma cells, relatively inefficient processes. A limited number of neutralizing targets have been identified in these studies, including the V3 loop (16, 17, 18), the CD4-binding site (CD4bs) (18, 19, 20, 21, 22), a conformational V2 epitope (23), a poorly defined epitope in gp120 (2G12) (15), and two epitopes in gp41, 2F5 (14, 16, 24) and 4E10 (25). In addition, two hu-mAbs have been described that are specific for conformational epitopes induced on binding of CD4 to gp120 and that neutralize several laboratory-adapted isolates (26). Phage display of recombinant Fabs derived from bone marrow cells of infected patients has mainly resulted in the isolation of hu-mAbs directed against the CD4bs (27, 28, 29). The most potent and cross-reactive of these is IgGb12, which is directed against a unique gp120 epitope that overlaps the CD4bs and the V2 domain (12, 30). However, the technical difficulties of this method have limited its application.

The recent availability of transgenic strains of mice expressing human Ig genes provides an alternative method for isolating hu-mAbs against HIV proteins. XenoMouse strains of mice were engineered by functionally inactivating the murine heavy chain and {kappa} light chain Ig loci and incorporating megabase-sized inserts of human DNA carrying Ig heavy chain and {kappa} light chain loci that express the majority of the human Ab repertoire (31). These animals produce a diverse array of fully human Abs that are suitable for therapeutic applications after immunization with a number of Ags (31, 32, 33, 34, 35, 36). In this report, a XenoMouse strain producing IgG2{kappa} Abs was tested for the ability to generate mAbs (designated Xeno-mAbs, to distinguish them from hu-mAbs isolated from HIV-1-infected patients by traditional methods) in response to immunization with a recombinant gp120 derived from the R5 primary isolate HIVSF162. Neutralizing responses against the autologous virus were readily induced, and hybridomas producing Xeno-mAbs reactive with rgp120 were efficiently isolated. The diversity of the Xeno-mAbs obtained, including some with potent neutralization activity against HIVSF162, and unique characteristics of the epitopes seen by these Abs, are described.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant proteins and synthetic peptides

Soluble, rgp120s from the R5 clade B primary isolates HIVSF162 (37) and HIVJR-FL (38) were secreted from HEK293 (39) cell lines stably expressing the recombinant protein from pcDNA3.1/Zeo(-) (Invitrogen, San Diego, CA). DNAs encoding these gp120s were prepared by PCR from the molecular clones and fully sequenced; the sequence for rgp120JR-FL was optimized at its initiation codon (40) and had a hexahistidine affinity tag embedded in a run of alanine and glycine residues at its C terminus. Soluble rgp120s were purified to >95% purity from cell culture medium by lectin chromatography using Galanthus nivalis snowdrop agglutinin (Sigma-Aldrich, St. Louis, MO) as previously described (41) and appeared to largely retain native conformation as determined by reactivity with soluble CD4 (sCD4) and mAbs against conformational epitopes in V2 and the CD4bs. Other soluble rgp120s were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. These included gp120s derived from the X4 clade B laboratory-adapted isolates HIVSF2 (no. 386), HIVIIIB (no. 3926) and HIVMN (no. 3927); the R5 clade B primary isolate HIVBaL (no. 4961); the R5 clade E primary isolate HIVCM235 (no. 2968); and the clade E primary isolate HIV93TH975 (no. 3234).

Expression and purification of fusion proteins carrying HIV-1 variable domains attached to the C terminus of an N-terminal fragment of a murine leukemia virus SU have been described (42). Wild-type and linearized V3JR-CSF fusion proteins and a fusion protein expressing the V1/V2SF162 domain (see Fig. 3Go for the region included) were used in this work.



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  		FIGURE 3. Mapping of epitopes in V1 and V2 domains. Xeno-mAbs previously scored reactive with the V1/V2SF162 fusion protein were retested against this Ag and three synthetic peptides. A, ELISA reactivities; B, sequences of the Ags. The sequence in the fusion protein (FP) corresponds exactly to the HIVSF162 isolate, and includes the stem that connects the V1/V2 domain to the core of gp120. The V1 peptides correspond to the HIVSF162 sequence, except that in P130-1 there is a serine in place of the cysteine N-terminal to the V1 loop, and P130-2 is missing an arginine that joins the V1 and conserved central region in the gp120 sequence. The V2 peptide (T15K) corresponds to the sequence of the HIVCaseA2 isolate; two residues that differ from the SF162 sequence are underlined.

 
Synthetic peptides T15K, P130-1, and P130-2 were purchased from Biosynthesis (Lewisville, TX). Peptides corresponding to various regions of the V3 loop from HIVMN (full length linear (no. 1840); full length circular (no. 1841); 1–20 (no. 1985); 11–21 (no. 1986); 21–40 (no. 1987)), HIVIIIB (no. 1590), and chimeric sequence PNDMN/IIIB (6–27 +QR, +C (no. 864)) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.

Immunization and hybridoma isolation

XenoMouse G2 animals (33), transgenic mice producing human {gamma}2{kappa} Abs, were immunized intradermally with rgp120SF162. Protein (20 µg) in the presence of Ribi adjuvant MPL + TDM (monophosphoryl Lipid A + synthetic trehalose dicorynomycolate) (Ribi ImmunoChem Research, Hamilton, MT) was used to prime each animal, and 15 µg of protein mixed with the same adjuvant was used to boost three times at 4-wk intervals. A final injection of 15 µg rgp120SF162 without adjuvant was given i.p. 4 days before fusion. In one experiment, immunizations were done with rgp120 that had been enzymatically deglycosylated by treatment with peptide-N-glycosidase F (New England Biolabs, Beverly, MA).

Splenocytes from immunized mice were harvested and fused with SP2/0 myeloma cells using standard techniques. Cell culture supernatants from wells containing hybridoma colonies were screened by ELISA against rgp120SF162, and cells from positive wells were expanded and retested. Cultures that remained positive were subcloned to generate stable, clonal hybridoma cell lines expressing Xeno-mAbs reactive with rgp120SF162. Xeno-mAbs and control human and murine mAbs were purified using protein A columns (Amersham Pharmacia Biotech, Piscataway, NJ) according to protocols supplied by the manufacturer.

Screening assays for mAb isolation and characterization

Hybridoma supernatants were screened by ELISAs as previously described (43), using alkaline phosphatase-conjugated goat anti-human IgG (Zymed Laboratories, San Francisco, CA) as the secondary Ab. For binding inhibition studies, sCD4 and mAbs at 1 mg/ml were biotinylated for 4 h at room temperature with 1/8 volume of biotinamidocaproate N-hydroxysuccinimide ester (1 mg/ml in DMSO; Sigma-Aldrich) followed by dialysis against PBS. Biotinylated probes and unlabeled competing reagents were mixed before adding to Ag-coated ELISA plates that were processed using streptavidin-AP (Zymed Laboratories) as the secondary reagent. Each biotinylated reagent was used at a concentration within its linear response range.

Measurement of HIV neutralization activity

Neutralization activity was determined with a single cycle infectivity assay using virions carrying Env-defective, luciferase-expressing HIVNL4–3 genomes (44) that were pseudotyped with HIVSF162 Env as previously described (42). Infections were conducted in a 96-well format, and luciferase activity was determined 48–72 h postinfection with a microtiter plate luminometer (Dynex Technologies, Chantilly, VA) using assay reagents from Promega.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Efficient induction of a gp120-specific humoral response in XenoMouseG2 mice

XenoMouse animals of the G2 strain produced rapid humoral responses against soluble HIV-1 gp120. Fig. 1GoA presents a typical profile of the humoral response of four XenoMouse G2 animals immunized with soluble recombinant gp120SF162 in the presence of Ribi adjuvant. All four animals produced detectable gp120-specific Abs after the first boost, and their Ab titers increased with subsequent immunizations. Sera of XenoMouse animals immunized with this protocol often contained neutralizing activity against the autologous HIVSF162 virus. Fig. 1GoB shows results of a neutralization assay performed with a preimmune serum and sera of three XenoMouse animals immunized with this protocol. The preimmune serum possessed no neutralizing activity, whereas two of three immune sera neutralized HIVSF162 with a 50% neutralizing dose (ND50) at ~1/25 dilution.



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  		FIGURE 1. Response of XenoMouse animals to rgp120. A, Xenomouse sera were assayed for reactivity with rgp120SF162 by ELISA at a dilution of 1/100. Sera of immunized animals were taken 3 days after the indicated boost with rgp120SF162. B, The ability of XenoMouse sera to neutralizae HIVSF162 was determined after the third boost with rgp120SF162. Sera were diluted 1/25 for the assays shown.

 
Isolation and initial characterization of gp120-specific Xeno-mAbs

Splenocytes from immunized XenoMouse animals fused efficiently with Sp2/0 myeloma cells, allowing the isolation of large numbers of gp120-specific hybridomas. These were initially screened by ELISA against the rgp120SF162 immunogen, and positive wells were subcloned and rescreened for reactivity. Single-cell clones obtained from positive subclones were then tested by ELISA for reactivity with fusion proteins expressing the gp120 V1/V2 and V3 variable domains (45), and with rgp120SF162 reduced with DTT to obtain preliminary mapping of the epitope specificities of the mAbs produced. Representative data are presented in Fig. 2Go. Epitopes seen by the Xeno-mAbs included sites both within and outside of the three variable domains tested. Eleven Xeno-mAbs were directed against the V1/V2 domain, and four were specific for the V3 domain. The Xeno-mAbs specific for these variable domains recognized linear epitopes, as defined by their similar reactivities with native and reduced rgp120SF162 (Fig. 2Go) and reactivity with synthetic peptides (see below). Of 20 Xeno-mAbs directed to gp120 sites outside the 2 major variable regions, only 3 reacted with reduced rgp120, indicating that the large majority recognized disulfide-dependent conformational epitopes. More precise definition of these epitopes is described below.



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  		FIGURE 2. Initial mapping of epitopes seen by Xeno-mAbs. ELISA reactivities of Xeno-mAbs were determined at 10 µg/ml against rgp120SF162 before and after reduction with DTT and against fusion proteins expressing the V1/V2 region of HIVSF162 or the V3 region of the closely related HIVJR-CSF. Xeno-mAbs are grouped by epitope class, as determined by additional experiments.

 
Characterization of Xeno-mAbs directed against V1/V2 epitopes

The reactivity of the V1/V2-specific Xeno-mAbs with reduced rgp120SF162 suggested that their epitopes might be mapped with synthetic peptides. A 17-mer peptide matching the N-terminal region of the V2 domain (corresponding to the HIVCaseA2 isolate (46), which differs from the HIVSF162 immunogen at two positions) was available, and 2 overlapping 15-mer peptides matching the HIVSF162 V1 domain and part of the adjoining conserved central sequence were synthesized (Fig. 3GoB). Ten of the V1/V2-reactive Xeno-mAbs reacted with the C-terminal V1 peptide (130-2) but not with the N-terminal V1 peptide (130-1) (Fig. 3GoA). Several of these Abs reacted only weakly with the peptide but were convincingly mapped to this region by the demonstration that their reactivity with the V1/V2SF162 fusion protein and with rgp120SF162 was efficiently blocked by the 130-2 V1 peptide (data not shown). Peptide 130-2 overlapped peptide 130-1 by seven residues and contained four additional V1 residues (KEMD) joined to the initial four residues of the conserved central region (GEIK), but was missing the arginine present between these sequences in SF162 gp120 (Fig. 3GoB). This suggested that a part or all of the KEMD sequence at the end of the V1 domain was critical for these epitopes.

The remaining V1/V2-reactive Xeno-mAb, 8.22.2, reacted with the V2 peptide, but not with either of the V1 peptides. The general region corresponding to the V2 peptide recognized by 8.22.2 has previously been shown to contain epitopes recognized by several neutralizing rat mAbs (47) and to be part of the epitope of a very potently neutralizing chimpanzee mAb, C108G (48). However, 8.22.2 appeared to recognize different V2 amino acids than did the previously described Abs. Those mAbs were localized to the N-terminal half of the peptide and were highly type specific for the HXB-2/HXB-10 sequences (C108G also recognized the BaL sequence (49)). The insensitivity of 8.22.2 binding to variations at two positions in the N-terminal region of T15K suggested that the 8.22.2 epitope was centered in the C-terminal half of this peptide. This is a relatively conserved region, consistent with the broad cross-reactivity of this Ab within clade B gp120s (see Table VGo, below).


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  		Table V. Reactivity of Xeno-mAbs with diverse rgp120s1

 
Competition assays were performed to obtain information about the proximity of the epitopes recognized by the Xeno-mAbs with each other and with previously described V2 epitopes. Two of the anti-V1 Xeno-mAbs and a previously described hu-mAb against a conformational epitope in V2 (697D) (23) were biotinylated, and the ability of various mAbs to block their binding to rgp120SF162 was determined (Table IGo). None of the V1 or V2 reactive mAbs was effective at blocking the binding of sCD4, whereas the control hu-mAb, 5145A, directed against an epitope that overlaps the CD4bs, was highly effective. As expected, neither 5145A nor 8.27.3, a Xeno-mAb directed against the V3 domain, blocked binding by any of the V1- or V2-specific mAbs. Thus, these V1 and V2 epitopes are not proximal to either the CD4bs or the V3 loop. All of the V1-specific Xeno-mAbs competed with both biotinylated V1-specific Abs, consistent with the peptide binding data localizing the epitopes of all of these Abs to the C terminus of the V1 domain. Neither of the V1-specific Xeno-mAbs was competed by 8.22.2, the Xeno-mAb directed against a linear V2 epitope, nor by two mAbs previously mapped to conformational V2 epitopes, the mouse mAb SC258 (50) and the hu-mAb 697D (23). Binding of biotinylated 697D was efficiently blocked by 8.22.2 and the murine anti-V2 mAb SC258, but not by any of the V1-specific Xeno-mAbs. Thus, in the three-dimensional structure of gp120, the linear V2 epitope is located in close proximity to the conformational V2 epitopes but not to the V1 epitopes, despite the proximity of the V1 and V2 peptides in the primary sequence.


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  		Table I. Mapping of V1 and V2 epitopes by binding competition assays1

 
Characterization of Xeno-mAbs directed against epitopes in V3

Four of the Xeno-mAbs were mapped to the V3 domain based on their reactivity with the V3JR-CSF fusion protein. The epitopes of these Xeno-mAbs were further characterized by ELISA against a series of peptides corresponding to regions of the V3 domain of HIVJR-CSF, HIVMN, and HIVIIIB gp120s. This allowed separation of the Xeno-mAbs into two discrete groups (A and B) that were distinct from three other groups (C, D, and E) defined by a panel of standard hu-mAbs isolated from HIV-1-infected human patients (Tables IIGo and IIIGo). Whereas all of the standard hu-mAbs reacted with the HIVMN 1–20 peptide, corresponding to the N-terminal region and the crown (residues 15–18) of the V3 loop, none of the Xeno-mAbs recognized this peptide. The group A Xeno-mAbs reacted with HIVMN peptide 11–30, implicating residues 21–30 in their epitopes. Their failure to react with HIVMN peptides 1–20 and 21–40 suggested that their epitopes spanned residue 20, near the crown of the loop. The reactivity of these Abs with the PNDMN/IIIB peptide, which contained the QR dipeptide characteristic of the V3IIIB sequence inserted in the MN sequence between residues 14 and 15, further suggested that the group A epitopes are not sensitive to residues N-terminal to the GPGR sequence.


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  		Table II. Mapping of V3 epitopes1

 

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  		Table III. Sequences of the V3 loop of HIVSF162 and the Ags used in Table IIGo

 
The Group B Xeno-mAb, 8.27.3, was distinguished from the others by its reactivity only with peptides expressing the full length V3 domain. This suggests that it recognized a discontinuous or conformational epitope. Its reactivity with both the linear HIVMN peptide and the linear form of the V3JR-CSF fusion protein indicated that the conformation of the 8.27.3 epitope was not dependent on the disulfide bond at the base of the V3 loop. This was the most potent neutralizing Xeno-mAb isolated (see below), indicating the functional relevance of the 8.27.3 epitope.

Characterization of Xeno-mAb epitopes outside the variable domains

Most of the Xeno-mAbs isolated did not react with either the V1/V2 or V3 variable region probes. To map these epitopes, binding competition assays were performed to measure the ability of each Xeno-mAb to inhibit binding of biotinylated Xeno-mAbs or biotinylated sCD4 to rgp120SF162 (Table IVGo). Six Xeno-mAbs and a control hu-mAb (5145a) efficiently blocked binding of sCD4 to rgp120SF162, indicating that they were directed against an epitope or epitopes overlapping the CD4bs of gp120. These Xeno-mAbs all recognized a disulfide bond-dependent epitope (Conf A) (Fig. 2Go), consistent with the conformational nature of the CD4bs and standard epitopes that mediate inhibition of sCD4 binding (51).


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  		Table IV. Mapping of epitopes in conserved domains by binding competition1

 
Eleven of the Xeno-mAbs directed against disulfide bond-dependent epitopes did not inhibit binding of sCD4 to rgp120SF162. All of these mAbs blocked the binding of one member of the group, 63G3/E2, to rgp120SF162, but did not inhibit binding of 38G3/A9, one of the Xeno-mAbs directed against the Conf A conformational epitope associated with the CD4bs (Table IVGo). These Xeno-mAbs therefore constituted a distinct competition group (Conf B). Two of these Xeno-mAbs inhibited 63G3/E2 only partially, which might reflect either lower affinity or reactivity with an epitope that only partially overlapped the other Conf B epitopes. The Xeno-mAbs to the Conf B epitopes all increased binding to rgp120SF162 of a Conf A Ab, 38G3/A9, and the three Conf A Abs tested all increased binding of a Conf B Xeno-mAb, 64G3/E2 (reflected in a negative % inhibition), suggesting possible allosteric effects induced by Abs to these sites.

The three Xeno-mAbs reactive with reduced rgp120SF162 but not with the V1/V2 or V3 fusion proteins constituted a third competition group (gp120 C). Each of these Xeno-mAbs inhibited 97B1/E8 binding, but did not significantly block binding by sCD4 or Xeno-mAbs directed against CD4bs or Conf B epitopes. The Xeno-mAbs directed against gp120 C epitopes were all isolated from mice immunized with rgp120SF162 that had been deglycosylated with peptide-N-glycosidase F. The binding of these Abs to rgp120SF162 was enhanced upon reduction of disulfide bonds (Fig. 2Go), suggesting that their epitopes are exposed by partial denaturation of the glycosylated molecules.

Extent of conservation of epitopes recognized by Xeno-mAbs

The degree of cross-reactivity of the Xeno-mAbs was explored by performing ELISA against a panel of eight rgp120s derived from three R5 clade B isolates, three X4 clade B viruses and two clade E isolates (Table VGo). The V1-specific Xeno-mAbs were all highly specific for rgp120SF162, consistent with this domain being the most highly variable in region in gp120 (52). The V2-specific Xeno-mAb, 8.22.2, reacted with all three R5 clade B rgp120s but with none of the X4 clade B rgp120s, consistent with both the existence of regions of significant sequence similarity (52) and the presence of determinants of tropism within this variable domain (53, 54, 55, 56). The V3-specific Xeno-mAbs recognized four or five rgp120s within clade B with no obvious bias with respect to coreceptor usage.

The Xeno-mAbs directed against epitopes outside of the variable domains were highly cross-reactive. Four of the CD4bs-specific Xeno-mAbs recognized all six of the clade B rgp120s, one recognized five, while one (the only one derived from immunization with deglycosylated rgp120SF162) was type-specific for HIVSF162. The Conf B Xeno-mAbs reacted with three to all eight rgp120s, in all but two cases including at least one of the clade E proteins. The gp120 C Xeno-mAbs were also cross-reactive, recognizing three to six of the six clade B rgp120s. The variation in recognition patterns of Abs within most of these groupings suggested that these Xeno-mAbs identified multiple epitopes in each of these epitope clusters.

Neutralizing activity of Xeno-mAbs

Each of the Xeno-mAbs was tested for the ability to neutralize the autologous HIVSF162 virus. A single cycle infection assay was used that employs virions bearing HIVSF162 envelope proteins and carrying a defective HIV-1 genome that expresses luciferase. Neutralization was seen for at least one of the Xeno-mAbs directed against each of four epitope clusters, the V1, V2 and V3 variable domains and the CD4bs (Fig. 4Go). None of the Xeno-mAbs against the Conf B domain or the linear gp120 C domain possessed neutralizing activity, even at 200 µg/ml (Table VIGo). This lack of neutralization may reflect either a lack of exposure of these domains in intact virions, or the lack of a function for these regions that can be interfered with by Ab binding.



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  		FIGURE 4. Xeno-mAb neutralization of HIVSF162. Representative neutralization assays of Xeno-mAbs ({diamond}, {square}, and {triangleup}) and hu-mAbs (•) against HIVNL4–3-luc virus pseudotyped with HIVSF162 envelope proteins, comparing V1 and V2-specific mAbs (A), CD4bs-specific mAbs (B), and V3-specific mAbs (C).

 

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  		Table VI. Xeno-mAb neutralization activity against HIVSF1621

 
The anti-V1 Xeno-mAbs all possessed potent neutralizing activity for HIVSF162, with ND50s ranging from below 0.3 µg/ml to 4.5 µg/ml (Table VIGo). The V2-specific Xeno-mAb, 8.22.2, had less potent neutralizing activity, with an ND50 of 48 µg/ml, which was however more potent than the control anti-V2 hu-mAb, 697D, which had an ND50 of 80 µg/ml. The V3-specific Xeno-mAbs varied widely in their neutralizing potency. Xeno-mAb 8.27.3 had the strongest neutralizing activity of all the Xeno-mAbs, with an ND50 of 0.1 µg/ml, while 8E11/A8 had an ND50 of 2.6 µg/ml. However, two additional V3-specific Xeno-mAbs with the same reactivity pattern as 8E11/A8, 6.1 and 6.7, had no detectable neutralizing activity at a concentration of 50 µg/ml. Four of the Xeno-mAbs directed against epitopes in the CD4bs also possessed moderate neutralizing activity, with ND50s in the range of 30–60 µg/ml, whereas two other Xeno-mAbs against this domain did not neutralize at 200 µg/ml. The variability in neutralization potency of the Abs directed against these neutralization domains may be due to different affinities or to subtle differences in the structure and functional roles of their epitopes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Identification of epitopes that mediate neutralization of primary HIV-1 viruses remains an important goal in HIV vaccine development. A major impediment has been the difficulty of isolating hu-mAbs by the standard methods of EBV transformation or phage display. This report demonstrates that XenoMouse strains can be used to efficiently isolate large panels of hu-mAbs directed against the known antigenic domains of HIV-1 gp120, and more importantly, against previously undescribed epitopes. After immunization with rgp120SF162 in Ribi adjuvant, neutralizing Xeno-mAbs were isolated that were directed against epitopes in the conserved CD4bs, and in the variable V1, V2, and V3 domains. Although neutralization determinants in most of these domains have been previously described, it was striking that the gp120 variable region neutralization epitopes that were identified by the Xeno-mAbs characterized in this study were distinct from those recognized by previously described mAbs.

The hypervariable V1 loop of gp120 was an immunodominant region for rgp120SF162, and all Abs directed against this domain had potent type-specific neutralizing activity. This is the first description of mAbs against the V1 domain (57). A previous study examining the humoral response of three laboratory workers infected with the laboratory-adapted X4 HIVIIIB virus reported that the V1 region was an immunodominant target of neutralizing Abs against the infecting strain (58), consistent with the results of the current study. The extremely high diversity of the V1 domain and the resulting type specificity of Abs against this region probably accounts for the previous failures to isolate V1-specific hu-mAbs. The relatively potent neutralizing activities of the anti-V1 Xeno-mAbs described in this study demonstrate that this region is a potent neutralizing target in at least one R5 virus, suggesting that such Abs may be an important component of the in vivo neutralizing humoral response against autologous virus. This would be consistent with immune selection driving the hypervariability of the V1 domain. These results also suggest that anti-V1 Abs may account, at least in part, for the potent neutralization of SHIV162P4 induced by DNA and protein immunization with rgp120SF162{Delta}V2 (59). The high diversity of the V1 domain and strict type specificity of Abs directed against this region, however, limit the utility of V1 as a vaccine target.

Although only a single Xeno-mAb directed against the V2 domain (8.22.2) was isolated in this study, this Ab was directed against a unique and interesting epitope. Unlike previously described mAbs against linear epitopes in V2 (47, 60), 8.22.2 was moderately cross-reactive, recognizing all three clade B R5 rgp120s tested. Other cross-reactive mAbs directed against V2 have been described, but those are directed against conformational epitopes that depend on the disulfide-bonded structure of the domain (23, 61, 62). Furthermore, 8.22.2 had more potent neutralizing activity against the R5 HIVSF162 isolate than 697D, the V2-directed hu-mAb previously reported to neutralize primary virus isolates (23). The neutralizing activity of 8.22.2 was consistent with the high potency for the primary HIVBaL isolate of the chimp mAb C108G (49), which is directed against a glycan-dependent epitope localized in the same region of V2.

Also of interest was the repertoire of V3 epitopes identified in this study. First, the V3-reactive Xeno-mAbs were quite cross-reactive, with the more potent of the two neutralizing Xeno-mAbs (group B) recognizing five of the six clade B rgp120s tested, and the other neutralizing V3-specific Xeno-mAb (group A) recognizing four of the six clade B rgp120s, including all three derived from primary isolates. The clade B rgp120 not recognized by either group was from the HIV-1IIIB isolate, which has an immunologically distinct V3 domain. The potently neutralizing group B Xeno-mAb, 8.27.1, was also unique in that it reacted with only full length V3 loop peptides, but not with overlapping 20-mers, suggesting that its epitope was more conformational than those recognized by previously described hu-mAbs. These epitope differences may result in part from differences in the immune repertoire between the XenoMouse strain used and humans. Another possibility is that the differences in V3 epitopes recognized by XenoMouse and traditional hu-mAbs resulted from the nature of the immunogen and/or the choice of screening Ag. In this study, R5 rgp120SF162 was used as both the immunogen and the screening Ag, whereas previously described V3-reactive hu-mAbs isolated from HIV-1-seropositive humans were screened against either rgp120HXB2 or V3MN peptides, both derived from laboratory-adapted X4 isolates (18, 63, 64). The unusual immunoreactivity of the V3HXB2 loop would not have allowed isolation of hu-mAbs reactive with the V3 epitopes identified in this study, and screening against HIVMN peptides may have introduced a bias toward reactivity with particular linear HIVMN sequences. Furthermore, the other rgp120 not recognized by the group A Xeno-mAbs was from HIVSF2. In a recent study, HIVSF2 was unusually resistant to V3-directed neutralizing Abs affinity purified from human patient sera (42). This suggests that the group A epitopes may actually be representative of neutralizing V3 targets seen in infected patients.

The majority of the Xeno-mAbs isolated in this study were directed against epitopes not contained within the V1, V2, or V3 variable domains. Except for those induced by immunization with deglycosylated rgp120SF162, these Abs were directed against conserved conformational epitopes that were separated by binding competition studies into two groups, one of which corresponded to the previously described CD4bs cluster (19, 20, 22, 51, 65, 66). Neither of these groups overlapped with the Xeno-mAbs against reduction-insensitive epitopes, which were preferentially presented by denatured rgp120. Most of the Xeno-mAbs against CD4bs epitopes had moderate neutralization activity, whereas none of the Xeno-mAbs against the other cluster of conformational epitopes neutralized HIVSF162. It is believed that one face of gp120 is occluded in the native trimeric Env complex (67, 68, 69), and it is possible that the Conf B epitopes were on this surface.

The present study demonstrated the utility of the XenoMouse system in generating interesting mAbs upon immunization with a soluble rgp120. It is likely that the range and nature of epitopes recognized can be expanded by the use of other forms of HIV-1 immunogens. Ags consisting of trimeric Env complexes, either soluble or membrane associated, may be effective immunogens for neutralization targets that are poorly expressed, if at all, on the gp120 monomer, and their use as screening agents may minimize the isolation of mAbs against the occluded face of gp120. The recently described stabilized trimeric forms of HIV-1 Env proteins (70, 71) may be particularly useful for this purpose. rgp120 immunogens derived from other isolates may induce responses against different classes of conserved and variable region epitopes. Furthermore, the use of a functional screening method, such as a direct screen for neutralization activity, may allow the isolation of more effective neutralizing Xeno-mAbs against already identified domains as well as novel targets.

A particularly intriguing aspect of the XenoMouse system is that it provides a unique opportunity to generate hu-mAbs against normal human proteins that are involved in infection by HIV-1. Promising targets for this approach include the HIV receptors, CD4, CXCR4, and CCR5 (72), and DC-SIGN, a dendritic cell surface protein that facilitates the transport of HIV-1 to secondary lymphoid organs, leading to enhanced infection of T4 cells (73). Because the receptor sequences are invariant, Xeno-mAbs raised against these Ags that possess appropriate specificities and affinities could theoretically inhibit infection by all strains of HIV-1.

As demonstrated in this study, the XenoMouse system provides a useful approach for isolating hu-mAbs against HIV-1 Env. The availability of transgenic mice that produce fully human Abs, together with the development of novel immunogens and functional screening assays, should facilitate the more complete mapping of targets for the neutralization of HIV-1, as well as the isolation of hu-mAbs with potential clinical utility as immunotherapeutic agents against this virus.


   Acknowledgments
 
We thank M. Gorny and S. Zolla-Pazner for providing a number of the hu-mAbs used in this study.


   Footnotes
 
1 This study was supported by U.S. Public Health Service Grants AI46283 and AI50452, by a grant from Abgenix, and by National Institutes of Health Training Grant T32-A107382 (to C.P.K.). Back

2 Address correspondence and reprint requests to Dr. Abraham Pinter, Public Health Research Institute, 225 Warren Street, Newark, NJ 07103-3535. E-mail address: pinter{at}phri.org Back

3 Abbreviations used in this paper: hu-MAbs, human mAbs; CD4bs, CD4-binding site; h, human; sCD4, soluble CD4; ND50, 50% neutralizing dose. Back

Received for publication February 25, 2002. Accepted for publication May 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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