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Journal of Virology, May 2009, p. 5087-5100, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.00184-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Broad Neutralization of Human Immunodeficiency Virus Type 1 (HIV-1) Elicited from Human Rhinoviruses That Display the HIV-1 gp41 ELDKWA Epitope{triangledown} ,{dagger}

Gail Ferstandig Arnold,1,2,3* Paola K. Velasco,1,2,3 Andrew K. Holmes,1,2,3 Terri Wrin,4 Sheila C. Geisler,1,2 Pham Phung,4 Yu Tian,2,{ddagger} Dawn A. Resnick,1,2 Xuejun Ma,1,2 Thomas M. Mariano,1,2 Christos J. Petropoulos,4 John W. Taylor,2 Hermann Katinger,5 and Eddy Arnold1,2,3*

Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey,1 Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey,2 Rutgers University—University of Medicine and Dentistry of New Jersey Molecular Biosciences Graduate Program, Piscataway, New Jersey,3 Monogram Biosciences, Inc., South San Francisco, California,4 Institute of Applied Microbiology, University of Agriculture, Vienna, Austria5

Received 26 January 2009/ Accepted 23 February 2009


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ABSTRACT
 
In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.


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INTRODUCTION
 
The development of an AIDS vaccine is an ongoing and urgent challenge. One of the major hurdles is that the specific correlates of protection against human immunodeficiency virus (HIV) are still largely unknown. Nonetheless, most agree that the full complement of cellular and humoral components of the immune system will be needed to combat this virus. This is especially true given that the virus resides permanently in its host, infects the very cells needed to direct effective immune responses, and evades the immune system, either by changing in appearance or hiding in subcellular compartments.

A broadly reactive neutralizing antibody response is likely to be critical as a first line of defense upon initial HIV exposure by aiding in the clearance of cell-free virions, targeting infected cells for destruction, and preventing viral spread through cell-to-cell transmission. The presence of inhibitory antibodies in highly exposed persistently seronegative individuals testifies to the importance of the humoral response (9, 37). Additionally, broadly neutralizing serum has been associated with healthier prognoses for infected individuals (27, 65) and may be vital for protecting offspring from their infected mothers (7, 79) and preventing superinfection by heterologous HIV strains (23, 84). Even if complete protection cannot be achieved by vaccine-derived antibodies, an early, well-poised and effective neutralizing antibody repertoire may be able to lower the set point of the viral load following the initial burst of viremia, an outcome that has been reported to translate into improved disease outcomes and reduced transmission of HIV (66, 74). Further benefits of neutralizing antibodies have been seen with passive immunization studies in macaques, in which administration of broadly neutralizing monoclonal antibodies (MAbs) has demonstrated that it is possible to provide protection from—and even sterilizing immunity against—HIV infection (5, 51, 66). There is also evidence that such antibodies may provide therapeutic benefits for chronically infected individuals, analogous to benefits realized with anti-HIV drug treatment regimens (87).

Despite the promising potential of broadly neutralizing MAbs, designing immunogens that can elicit such cross-reactive neutralizing responses against HIV has been a surprisingly difficult task. Since the majority of the host's B-cell response is directed against the envelope (Env) glycoproteins, gp120 and gp41, vaccine efforts have concentrated on these proteins and derivatives thereof in approaches ranging from the use of Env-based peptide cocktails to recombinant proteins and DNAs made with varied or consensus sequences and diverse, heterologous prime/protein boost regimens (reviewed in references 36, 58, and 70). These iterative studies have shown notable improvements in the potency and breadth of neutralizing responses induced. However, concerns exist regarding immunogens containing extraneous epitopes, as is the case with intact subunits of Env, and the nature of the immune responses they may elicit. A polyclonal burst of antibodies against a multitude of nonfunctional epitopes may include a predominance of antibodies that are (i) low affinity and/or nonfunctional (reviewed in reference 72); (ii) isolate specific (25); (iii) able to interfere with the neutralizing capabilities of otherwise-effective antibodies (via steric hindrance or by inducing various forms of B-cell pathology) (67); or (iv) directed against irrelevant epitopes instead of more conserved (and sometimes concealed) epitopes that might be able to elicit more potent and cross-reactive neutralizing responses (28, 71, 91).

We have developed a system that can be used to present essentially any chosen epitope in a stable, well-exposed manner on the surface of the cold-causing human rhinovirus (HRV). HRV is itself a powerful immunogen and is able to elicit T-cell as well as serum and mucosal B-cell responses (reviewed by Couch [22]) and has minimal immunologic similarity to HIV (data not shown). Chimeric viruses displaying optimal epitopes should be able to serve as valuable components in an effective vaccine cocktail or as part of a heterologous prime/boost protocol. We have shown previously that HRV chimeric viruses displaying HIV-1 gp120 V3 loop sequences are able to elicit neutralizing responses against HIV-1 (75, 82, 83).

In this study, we focused our attention on presenting part of the membrane-proximal external region (MPER) of the transmembrane glycoprotein gp41, a region of approximately 30 amino acids adjacent to the transmembrane domain (reviewed in references 59 and 97). The MPER plays an important role in the process of HIV fusion to the host cell membrane (60, 78). This region is also involved in binding to galactosylceramide, an important component of cell membranes, thus permitting CD4-independent transcytosis of the virus across epithelial cells at mucosal surfaces (1, 2). These functions likely explain this region's sequence conservation and the efficacy of antibodies directed against the MPER (97), particularly given that an estimated 80% of HIV-1 infections are sexually transmitted at mucosal membranes. In fact, potent responses against the MPER are associated with stronger and broader neutralizing capabilities in infected individuals (68). A conserved, contiguous sequence of the MPER, the ELDKWA epitope (HIV-1 HxB2 gp41 residues 662 to 668), is recognized by the particularly broadly neutralizing human MAb 2F5 (11, 62, 85) and is highly resistant to escape mutation in the presence of 2F5 (49). 2F5 was also used in the MAb cocktails reported to confer passive, protective immunity in macaques (5, 51). In addition, infected individuals producing neutralizing antibodies directed against the ELDKWA epitope have been seen to exhibit better health (16, 29), including persistent seronegativity (8), and reduced transmission of HIV to offspring (89). While none of the vaccine-induced immune responses generated against this region has been effective thus far (19, 24, 26, 33, 35, 38, 40, 42, 44-48, 50, 53, 54, 56, 57, 61, 63, 69, 93, 96) (see Table S1 in the supplemental material), more appropriate presentations of MPER epitopes should produce valuable immunogens that can contribute to a successful vaccine.

In this study, we have grafted the ELDKWA epitope onto a surface loop of HRV connected via linkers of variable lengths and sequences and selected for viruses well recognized and neutralized by MAb 2F5. In so doing, we have been able to create immunogens capable of eliciting antibodies whose activities mimic some of those of 2F5. The combinatorial libraries produced were designed to encode a large set of possible sequences and, hence, structures from which we could search for valuable conformations. This work illustrates that HRV chimeras have the potential to present selected HIV epitopes in a focused and immunogenic manner.


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MATERIALS AND METHODS
 
Cells, viruses, and media. H1-HeLa cells (20) were used for transfection and propagation of plasmid-derived HRV14 (43) and HRV14:ELDKWA chimeras. Media M and PA (75) with 1 to 10% fetal bovine serum were used for plaque isolation and propagation of viruses. Dulbecco's modified Eagle medium (Gibco-BRL) was used for cell transfection experiments. Escherichia coli DH10B cells (ElectroMAX; Gibco BRL) were used for electroporation of plasmid DNA.

Monoclonal antibodies. Human MAb 2F5, which binds the HIV-1 gp41 amino acid sequence ELDKWA (21, 73), was provided by Hermann Katinger. Murine MAb 17, which binds to the neutralizing immunogenic site I of HRV14 (81), was provided by Roland Rueckert (University of Wisconsin, Madison). Horseradish peroxidase (HRP)-conjugated goat anti-human immunoglobulin G (IgG), HRP-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-guinea pig IgG were from Cappel ICN (Irvine, CA).

Peptides. Biotin-conjugated LELDKWASL-NH2, acetyl (Ac)-LELDKWASL-NH2, Ac-EQELLELDKWASLW-NH2, and keyhole limpet hemocyanin (KLH)-conjugated C-LELDKWASL-NH2 and C-EQELLELDKWASLW-NH2 peptides were bought from NeoMPS, Inc. (formerly Multiple Peptide Systems, San Diego, CA).

Construction and generation of chimeric HRV14:ELDKWA libraries. The process for construction and generation of the chimeric HRV14:ELDKWA libraries is illustrated in Fig. S1 of the supplemental material. All combinations of N- and C-terminal DNA oligonucleotides encoding the library were hybridized in the ELDKWA-coding region and extended with Klenow DNA polymerase I (New England Biolabs, Beverly, MA) to generate double-stranded DNA cassettes. (Cross-contamination of a number of the DNA oligonucleotide stocks at this stage became apparent upon examination of the sequences of the resultant chimeras; this reduced the diversity of the library explored and was taken into account in analyzing the results.) These cassettes were digested with restriction endonucleases ClaI and ApaI and ligated into the corresponding ClaI- and ApaI-digested p3IIST plasmid (83). Mutagenized plasmids were electroporated into DH10B cells with a Gene Pulser (Gibco-BRL, Carlsbad, CA), under conditions specified by the manufacturer. Transformed cells were grown in bulk liquid cultures at 30°C, and plasmids were isolated using Qiagen MiniPrep spin kits (Valencia, CA). The resulting plasmid pools were used as templates for in vitro transcription reactions and their mRNAs were transfected into HeLa cells (82). Pulsed cells were plated with an equal number of unpulsed cells for virus propagation and harvesting. Chimeric viruses were harvested from cultures as previously described (75). The number of transfectants was determined by counting PFU from pulsed cells mixed with unpulsed cells.

Purification of virus libraries and viral isolates. The individual viruses and virus pools of combinatorial library II were purified according to the protocol described by Zhang et al. (95). In brief, clarified virus lysates were obtained by alternate freezing and thawing of concentrated suspensions of infected cells followed by centrifugation to remove cell wall debris. The clarified lysates were subjected to DNase I treatment followed by ultracentrifugation (using a 30% sucrose cushion in a Beckman 45 Ti rotor at 45,000 rpm for 2 h at 15°C) and resuspension of the viral pellets in 10 mM Tris-HCl, pH 7.4, 0.1 M NaCl. After this, a 7.5 to 45% sucrose density gradient fractionation step was used, followed by a subsequent ultracentrifugal pelleting and resuspension. An abbreviation of this protocol was used for the purification of the individual viruses and virus pools of combinatorial library III. Library III viruses were purified only through the first ultracentrifugation (partially purified) and, thus, included cell-derived ribosomes.

Enzyme-linked immunosorbent assays (ELISAs) of chimeric viruses. Ninety-six-well plates (Nunc, Rochester, NY) were coated with 0.1 µg/well of MAb 2F5 in 50 mM sodium borate, pH 8.5, and incubated overnight at 4°C. Plates were blocked with 3% gelatin in phosphate-buffered saline (PBS) at 37°C for 1 h. Plates were then washed six times with wash buffer (PBS containing 0.05% Tween 20). Sequential twofold dilutions of virus stock (either pools or single clones) were added and incubated for 2 h at 37°C. Plates were washed and 0.4 to 1.3 µg/ml of anti-HRV14 MAb17 was added, incubated for 1 h at 37°C, washed six times, and treated with HRP-conjugated goat anti-mouse IgG (at a 1:1,000 dilution). After a 1-h incubation and final washing, peroxidase substrate (0.3 mg/ml tetramethylbenzidine dissolved in 10% dimethyl sulfoxide and 0.18 M Na citrate, pH 3.95) was added. The reaction was catalyzed by the addition of H2O2 to 0.009%, allowed to develop color, and then stopped by the addition of an equal volume of 1 M H2SO4. Titers are expressed as reciprocals of virus stock dilutions (relative to an initial concentration of 1 x 108 PFU/ml) at which an optical density at 450 nm (OD450) of 0.5 was achieved.

Chimeric virus neutralization assays. Chimeric virus neutralization assays were performed as previously described (82). Briefly, 50 µl of 2 x 105 PFU/ml of chimeric viruses was incubated in M medium for 1 h at 34.5°C, 2.5% CO2 with 50 µl of sequential twofold dilutions of MAb 2F5 (starting at a 4-µg/ml concentration) in 96-well microtiter plates (Nunc, Rochester, NY). Fifty µl of H1-HeLa cells was added at a concentration of 2 x 105 cells/ml. The plates were incubated at 34.5°C, 2.5% CO2 for up to 48 h. Fifteen µl of a 5-mg/ml solution of 3-(4,5 dimethylthiazol)-2-yl-2,5-diphenyltetrazolium bromide (Sigma) in PBS was added to the wells. After a 1.5-h incubation at 34.5°C, 2.5% CO2, the reaction was stopped by adding 150 µl of 20% sodium dodecyl sulfate in 50% N,N-dimethylformamide. Cell survival was defined according to the percentage of the OD570 observed for cell control wells (corresponding to 100% viability). 2F5 50% inhibitory concentrations correspond to the 2F5 concentration necessary to neutralize chimeric virus infection by 50%.

Immunoselection of chimeric viruses bound by MAb 2F5. Immunoselection with no competition was performed as previously described (75, 82) with some modifications. Ninety-six-well Immunosorp plates (Nunc, Rochester, NY) were coated with MAb 2F5 (0.84 to 6.7 nM) (Fig. 1). After overnight incubation at 4°C, plates were blocked with 3% gelatin in PBS for 1 h at 37°C. Plates were washed six times with wash buffer (PBS containing 0.05% Tween 20) and 3 x 105 PFU/well of chimeric viruses was added and incubated for 2 h at 34.5°C and 2.5% CO2. Plates were washed five times with wash buffer and three times with PBS only. Antibody-bound virus was eluted with either 2 x 104 H1-HeLa cells/well or Ac-LELDKWASL-NH2 at 75 to 300 µM (Fig. 1). When cells were used for elution, plates were incubated at 34.5°C, 2.5% CO2 for up to 72 h (until cells reached 100% cytopathic effect), and virus was harvested as described previously (75). When peptide was used for elution, peptide addition was followed by incubation at 34.5°C, 2.5% CO2 for 2 h, after which eluted virus was propagated in 60-mm propagation plates seeded with 5 x 105 cells at 34.5°C, 2.5% CO2 and grown to 100% cytopathic effect. Competitive immunoselection was performed similarly, with one modification: prior to virus addition to the antibody-coated plates, virus was preincubated for 1 h at room temperature with Ac-LELDKWASL-NH2 (at 0.04 to 40 µM).


Figure 1
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  		FIG. 1. Immunoselection schemes used to enrich for chimeric viruses with optimal binding to immobilized MAb 2F5. Pools I, II, and III (consisting, respectively, of libraries 12, 14, 16, 42, 44, 46, and 64, libraries 14 and 44, and libraries 11, 12, 14, 16, 21, 22, 24, 26, 41, 44, 61, 62, and 64) were independently subjected to increasingly stringent binding conditions. (These pools were originally designed to consist of chimeras of differing insert sizes, but a few of the oligonucleotides used to generate the viruses were cross-contaminated at an early stage.) A, B, and C correspond to the first, second, and third rounds of selection, respectively. Information in the boxes indicates the following: 2F5, the concentration (nanomolar) of 2F5 used for the immobilization of viruses to 96-well plates; Pep, the concentration (micromolar) of Ac-LELDKWASL-NH2 peptide used for competition; elute, the concentration (micromolar) of Ac-LELDKWASL-NH2 peptide used for elution of bound chimeric viruses; large N, not subjected to competitive immunoselection. The default "eluent" was cells.

Plaque isolation and sequencing of chimeric viruses. Individual virus isolates were obtained by plaque isolation (82). Virus isolates were designated according to the number of biased residues in the N- and C-terminal linkers (1, 2, 4, or 6 for each terminus), the immunoselection round from which they were derived (A, B, or C), a possible indication of no peptide competition during immunoselection (N), the concentration of peptide used to compete with or elute chimeric virus (0 to 300 µM), and the clone number. Viral RNAs were isolated and reverse transcribed, and resultant cDNAs were amplified by standard PCR methods. The DNA fragments were then sequenced (Gene-Wiz, North Brunswick, NJ).

Immunization of guinea pigs. Three young male Dunkin Hartley guinea pigs (Cocalico Biologicals, Reamstown, PA) were immunized subcutaneously with each chosen chimeric virus. For the library II studies, approximately 50 µg of fully purified chimeric virus in 10 mM Tris-HCl, pH 7.4, 0.1 to 0.4 M NaCl mixed with an equal volume of Freund's complete adjuvant (CFA; first immunization) or incomplete Freund's adjuvant (IFA; boosts) was used for immunization according to the following schedule: week 0, initial virus immunization; week 4, first virus boost; week 9, second virus boost. Sera were collected at weeks –1, 7, and 12. For the library III studies, 50 to 100 µg of partially purified chimeric virus in 10 mM Tris-HCl, pH 7.4, 0.1 M NaCl without adjuvant was used for immunization according to the following schedule: week 0, initial virus immunization; week 4, first virus boost; week 9, second virus boost, except in the cases of guinea pigs 65 to 67 (which were given a half-dose of virus and a half-dose [40 µg] of a KLH-conjugated 14-mer peptide [C-EQELLELDKWASLW-NH2] with CFA) (see Fig. 5). In some cases, an additional peptide-only boost was given on week 13 using 80 µg of either the KLH-conjugated 14-mer peptide or a 9-mer peptide with IFA (C-LELDKWASL-NH2) (see Fig. 5). A peptide-only control was done using larger and more frequent doses. Three guinea pigs were immunized with 80 µg each of the KLH-conjugated 14-mer peptide at weeks 0, 4, and 8 (initially with CFA, then with IFA). Sera were collected by femoral bleeds or heart sticks at weeks –1, 7, 12, and 16 (when there was a 13-week peptide boost) for the virus-containing immunizations and weeks –1 and 10 for the peptide-only controls.


Figure 5
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  		FIG. 5. HRV14:HIV-1 gp41 ELDKWA virus-derived reciprocal serum neutralization titers at which there was 50% inhibition of HIV-1 pseudovirus replication (luciferase activity). Sera were obtained via immunization of three guinea pigs according to the following schedules: (i) for chimeric virus with or without peptide, week 0, initial immunization, ~50 µg virus, no adjuvant; week 4, first boost, ~50 µg virus, no adjuvant; week 9, second boost, either ~50 µg virus, no adjuvant, or ~25 µg virus plus 40 µg KLH-conjugated 14-mer peptide (EQELLELDKWASLW) with CFA; in some cases, week 13, no virus, 80 µg 14-mer peptide or 9-mer peptide (LELDKWASL) with IFA; (ii) for the peptide control, 80 µg 14-mer peptide at weeks 0 (with CFA), 4 (with IFA), and 8 (with IFA). (The peptide control immunization involved larger and more frequent doses of peptide than those used for any of the peptide-boosted virus immunizations.) Data in the figure correspond to sera collected 2 weeks after the final immunization. Serum codes with the same number are from the same animal; the number of Ps indicates the number of peptide boosts given before sample collection. The core insert sequence (E/A)LDKWA is shown in red. Residues marked in green appeared at the same relative positions in one or more HIV-1 isolates represented in the Los Alamos database in 2003; residues marked in black did not. The titers are averaged from ≥3 independent experiments with the exceptions of the IgG samples, the 147 to 149 series of sera, and the clade C tests. The same 67-PP serum sample was tested in its normal, heat-inactivated form and after protein A purification. A sampling of representative curves is shown in Fig. S2 of the supplemental material.

The use of animals for this research was in strict compliance with federal and institutional guidelines.

Protein A purification of serum IgG. Purified IgGs from some of the guinea pig sera were isolated using Sepharose beads bound with protein A (GE Healthcare). The manufacturer's recommended procedures were followed, after which Ultrafree concentrators (10,000 molecular weight cutoff; Millipore) were used for buffer exchange with Dulbecco's phosphate-buffered saline, pH 7.2. IgGs were assessed for purity via sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

HIV-1 pseudovirus neutralization assay. A recombinant pseudovirus assay developed at Monogram Biosciences, Inc. (South San Francisco, CA) was used to determine neutralization titers (76). In this assay, HIV-1 gp160 cDNAs generated from HIV-1 virus stocks were inserted into the pCXAS expression vector (under the control of the cytomegalovirus intermediate-early promoter-enhancer). These vectors were cotransfected with an HIV-1 genomic vector, containing the firefly luciferase reporter gene in place of the env gene, into HEK293 cells. The harvested recombinant viruses were preincubated with various dilutions of heat-inactivated guinea pig sera and then added to U87 cells expressing the CD4 receptor and the CCR5 and CXCR4 coreceptors. Neutralizing titers were calculated after one round of replication as the reciprocal of the guinea pig serum dilution yielding 50% inhibition of luciferase activity (compared with pooled normal guinea pig serum values). With only a few exceptions, each value shown in Fig. 5 was determined from at least three independent experiments.

ELISAs of guinea pig antisera and purified IgGs. Ninety-six-well Maxisorp plates (Nunc, Rochester, NY) were coated with 100 µl/well of 180-ng/ml unconjugated Ac-EQELLELDKWASLW-NH2 peptide in Dulbecco's phosphate-buffered saline, pH 7.2, lacking Ca2+ and Mg2+ (DPBS) and incubated overnight at 4°C. Plates were blocked with 175 µl/well of 5% fraction V bovine serum albumin in DPBS, pH 7.2, at 37°C for 1 h. Plates were then washed three times with 175 µl/well of wash/dilution buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.2, 0.05% Tween 20, 0.1% bovine serum albumin). Serial dilutions of guinea pig antisera or IgG or purified 2F5 MAb were added and incubated for 2 h at 37°C. Plates were washed three times and treated with 100 µl/well of HRP-conjugated goat anti-guinea pig IgG (at a 1:1,000 dilution, for serum and guinea pig IgG samples) or HRP-conjugated goat anti-human IgG (at a 1:2,000 dilution, for 2F5). After a 1-h incubation at 37°C and three washes with wash/dilution buffer and one with DPBS, peroxidase substrate (0.3 mg/ml tetramethylbenzidine in 0.18 M Na citrate, pH 3.95) was added. The reaction was catalyzed by the addition of H2O2 to 0.009%, allowed to develop color, and then stopped by the addition of an equal volume of 4 M H2SO4.


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RESULTS
 
Combinatorial library I. Three combinatorial libraries were constructed. The first library encoded the sequence LL(E78/A22)LDKW(A72/E13/T13/K2)(S83/N17)LW (reflecting the percentages of variable residues among HIV-1 isolates at the time of library design (Los Alamos Database [39]) flanked by zero to four randomized residues linking the epitope to the surface of HRV (see Table S2A in the supplemental material). The well-conserved amino-terminal LL and carboxy-terminal LW were included in an attempt to best present the epitope. The site chosen for display of the ELDKWA sequence was the major loop of the neutralizing immunogenic site II of HRV14, loop 2 of the so-called VP2 puff of VP2 (3, 77), used previously for the insertion of foreign sequences (4, 75, 82, 83).

ELDKWA-encoding chimeric HRV plasmids were constructed and used for the in vitro production of an RNA transcript library. After transfection of the RNA library into H1-HeLa cells, a pool of chimeric viruses was produced, containing approximately 6 x 103 independent viruses (measured as a function of the number of plaques obtained by transfection).

This chimeric virus pool was expected to contain a variety of ELDKWA presentations. To enrich for those viruses that displayed the HIV sequence in conformations most closely resembling those of the native 2F5 epitope (and possibly those most likely to elicit 2F5-like neutralizing antibodies), the chimeric virus pool was subjected to immunoselection with 2F5. Viral RNA from five recombinant viruses was sequenced, revealing each to be missing most or all of their HIV epitope (not shown). Nine recombinant plasmids were sequenced, with each encoding intact HIV core epitopes (not shown) and being used as templates for RNA synthesis, followed by transfection. All nine of the resulting viruses were found to have deletions in the region of the epitope (although one had an LLEL sequence). Rather than continue to search for intact recombinant viruses from this library, we designed a second library.

Combinatorial library II. The second library was designed assuming that the previously encoded hydrophobic LL and LW residues flanking the (E/A)LDKWAS core were unfavorable on the solvent-exposed surface of HRV. Thus, the sequence (L20/X80)(E78/A22)LDKWAS(L20/X80) was flanked by zero to four randomized residues on each side and inserted at the same site as that used for library I (X is any of the 20 amino acids) (see Table S2B in the supplemental material). A total of 2.9 x 106 independently transfected viruses were produced from this library.

Viruses with intact (E/A)LDKWA epitopes were observed in 11 of 12 viruses examined (one encoded ELDKWES); however, 5 of the 12 viruses sequenced lacked the encoded C-terminal L/X and C-terminal randomized residues (see Table S2B in the supplemental material), possibly because their presence reduced viral fitness. Only one of the 12 N-terminal L/X residues was an L, suggesting that this residue may have reduced virus viability. All of the 12 viruses contained N-terminal randomized residues. One HRV residue N-terminal to the linker was either mutated from an A to an S or deleted.

A surprisingly large percentage of N-terminal linker positions consisted of β-turn-forming residues (G, P, S, and T [18]) (Table 1), approximately twice that expected for both the randomized and L/X positions [considering that NN(G/C) was used as the codon and depending on whether the aforementioned HRV residue was mutated or deleted], suggesting that a β-turn may have helped accommodate the observed sequences in the context of HRV. Furthermore, there were more than twice as many helix-breaking residues (G, P, Y, or N [18]) as expected among the N-terminal L/X residues. On the C-terminal side of the epitope, the total number of linker residues was too small to note any significant patterns.


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  		TABLE 1. Sequence characteristics of linkers from library II and III chimeras

Following purification (to enrich for viruses that might be more easily purified at later stages), the library was subjected to immunoselection with MAb 2F5. 2F5 was immobilized to 96-well plates with one of four coating concentrations (1.0, 0.25, 0.1, and 0.025 µg/ml, corresponding to 67, 17, 6.7, and 1.7 nM, respectively). Four rounds of immunoselection were performed to include each of 16 combinations of 2F5 concentrations.

Sixteen chimeric viruses from rounds 2 to 4 were analyzed, revealing 15 unique sequences, all with intact (E/A)LDKWA inserts (see Table S2B in the supplemental material). Like the unselected viruses from this library, roughly half of the selected viruses lacked the encoded C-terminal randomized and L/X residues (in numbers too small for sequence pattern analysis) (Table 1). Similarly, there were only five N-terminal randomized residues; however, at the N-terminal L/X site, the preponderance of β-turn-forming or helix-breaking residues was even more extreme after immunoselection, with 3.4 and 5.3 times that expected, respectively, suggesting that recognition of the insert by 2F5 was improved by the conformational effects of these residues. In contrast, despite conservation of the N-terminal L among HIV-1 isolates, no L residues were encoded in this position among the chimeras. Notably, immunoselection resulted in a reduction of A residues at the (E/A) site from 33% (unselected set) to 13% (immunoselected set), consistent with the known preference of 2F5 for binding to ELDKWA versus ALDWKA (98).

A chimeric virus neutralization assay was used to see if 2F5 could neutralize chimeric virus infection of HeLa cells. A wide range of neutralization sensitivities was seen for the different chimeras (data not shown). We hypothesized that the viruses most sensitively neutralized by 2F5 presented their (E/A)LDKWA inserts most like HIV on average and might, therefore, be more capable of eliciting 2F5-like antibodies. Thus, the eight chimeric viruses most sensitively neutralized by 2F5 were individually purified and used to immunize three guinea pigs each. Sera were collected and tested for their ability to neutralize HIV-1 infection in cell culture using two assays, one utilizing human peripheral mononuclear cells (PBMC) activated by phytohemagglutinin (88), and another using single-round HIV-1 pseudovirus neutralization (76), measured by pseudovirus-driven luciferase activity. Some of the sera were able to modestly neutralize the HIV-1 IIIB and HIV-1 ALA laboratory strains grown in PBMC; however, none were able to neutralize any of the primary HIV-1 pseudoisolates tested (data not shown). Thus, a third library was designed.

Combinatorial library III. The third library encoded the core (E/A)LDKWA epitope flanked by larger linker sequences, including mixtures of randomized residues, HIV-1 residues, and cysteine residues (Fig. 2). The core immunogen was encoded to display 60% E and 40% A (the prevalence represented in the Los Alamos Database at the time of the library design). Four to 14 of 16 possible N- and C-terminal linker residues flanking the (E/A)LDKWA sequence were randomized (X) to encode any of the 20 amino acids. To maximize the degree of conformational diversity and to possibly assist in displaying the most relevant ELDKWA conformations available to the viruses, 2 to 12 of these residues were also biased (B), encoding 50% randomized residues and 50% residues normally found at these sites among HIV isolates. A cysteine residue was also added to each linker on the chance that a disulfide bond could form and, possibly, help stabilize the resultant immunogen-containing loop and/or protein in which it resided.


Figure 2
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  		FIG. 2. Design of ELDKWA library III and observed sequences. Subscript numbers correspond to relative percentages encoded (in the case of the HIV residue, reflecting the values among HIV-1 isolates at the times of the library design. The chimeric virus names reflect the number of biased residues on the N- and C-terminal sides of the ELDKWA insert, respectively (1, 2, 4, or 6), the immunoselection round (A, B, or C), the concentration of Ac-LELDKWASL-NH2 peptide used for competition (0 to 40 µM) or the possibility of no competitive peptide used for the immunoselection (N) but used for elution instead (75 to 300 µM), and the clone number. The N-terminal side of the insert of UN-14-26 has a deletion of at least a cysteine residue. Positions with a ? were unreadable and may comprise any of the 20 amino acids.

Part of the ELDKWA library was produced (with a diversity of 2.8 x 106 recombinant viruses) and divided into three pools (Fig. 1). Each pool was immunoselected in a variety of ways. Four different concentrations of 2F5 (0.1, 0.05, 0.025, and 0.0125 µg/ml, corresponding to 6.7, 3.4, 1.7, and 0.84 nM) were chosen for immobilization to 96-well plates, with subsequent rounds of immunoselection done using less 2F5/well (to select for more specific binding of the chimeric viruses to the 2F5). Under most conditions, competing Ac-LELDKWASL-NH2 peptide (0.02 to 40 µM) was added together with the chimeric viruses to the antibody-coated wells, also increasing stringency with progressive rounds of selection. In some cases, no competing peptide was added during the virus capture step but was instead added subsequently (increasing the concentration stepwise from 75 to 300 µM) to elute the captured viruses for harvest (versus otherwise adding HeLa cells to "elute" captured viruses for this step).

The different immunoselection schemes yielded 30 subpools (11 from pool I, 12 from pool II, and 7 from pool III). The subpools from which we isolated chimeras with conformationally relevant ELDKWA presentations were chosen based on ELISAs using immobilized 2F5 to capture pertinent viruses (Fig. 3). Subpools in which more members were captured generated higher OD450 values. An example of six subpools from pool II shows that the subpools with greatest average affinity to 2F5 were those that were (i) selected in the presence of the highest concentrations of competing Ac-LELDKWASL-NH2 peptide (cf. the B40 and B10 subpools [using 40 and 10 µM competing peptide] versus the B0.64 subpool [using 0.64 µM competing peptide]), and (ii) eluted from immunoselection plates using high peptide concentrations (cf. the BN300 subpool [eluting with 300 µM peptide] versus the BN75 subpool [eluting with 75 µM peptide]).


Figure 3
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  		FIG. 3. ELISA curves representing the effects of immunoselection on the binding of pools of chimeric viruses to immobilized MAb 2F5. In this example, the starting set was pool II. Subsets are designated with an A or B to indicate their derivation after one or two rounds of immunoselection, respectively. The presence of an N indicates that no competitive peptide was used. The subset numbers reflect the micromolar concentration of Ac-LELDKWASL-NH2 peptide used to compete with the chimeric viruses for binding to immobilized 2F5.

Four subpools were chosen as sources for individual virus isolates: one of the pool I C subpools, one each from two pool II B subpools (one eluted with cells and the other with peptide), and one of the peptide-eluted pool III A subpools (Fig. 1). Chimeric viruses were isolated and plaque purified twice from the preferred subpools. Individual chimeras were then characterized based on their sequences and their antigenic and immunogenic properties.

Assessment of virus sequences. An examination of the inserted sequences of 100 chimeras (Fig. 2) revealed a number of nonrandom trends. One trend showed that some of the DNA oligonucleotides used to construct the libraries had gotten mixed together. As a result, the chimeric virus sequence distribution was skewed, due to overrepresentation of faster-growing chimeras (generally those with shorter inserts) and was taken into account when the results were analyzed.

Beyond this technical limitation, the most prominent observation is that both linkers were found to be rich in residues known to promote β-turns, whether or not the viruses had been immunoselected (Table 1). The N-terminal linkers, particularly, were characterized by having more than three times the percentage of β-turn-promoting residues (G, P, S, and T) expected among both unselected and selected chimeras. While the percentage was lower for the C-terminal linker residues, there was still a doubling of the percentage of β-turn-promoting residues compared to that expected, also regardless of selection. The N-terminal linker residues also showed an abundance of helix-breaking residues (3.4 times more than expected overall). This trend was not observed on the C-terminal side of the insert. This observation is notable, as (i) the ELD of the ELDKWA epitope (92) and approximately 35 residues N terminal to the epitope have been seen, crystallographically, to be part of the six-helix bundle formed during the fusion process (17, 86, 92), (ii) comparable residues on the N-terminal side of a number of ELDKWA-based peptides assume helical structures in solution (12, 13), and (iii) none of the HIV-biased residues of the N linker promote β-turns, although there is a helix-breaking N residue encoded in this region. Therefore, the β-turn-forming propensity of the N linkers of this library (and library II) likely reflects their role in enhancing viability of the chimeric viruses, possibly involving coat protein folding, maturation by proteases, or interactions with biological partners.

In contrast to the linker residues, the presence of an E or A residue in the first amino acid position of the (E/A)LDKWA immunogen does appear to be sensitive to immunoselection, as was seen with library II. Unselected chimeras displayed 46% E and 45% A (as well as 4.4% each of V and G, apparently from contamination of the second base of the codon meant to encode only E and A). In contrast, chimeras selected with 2F5 displayed 58% E and 32% A (as well as 6.5% V and 3.2% G), in support of the observation that 2F5 binds preferably to E versus A (98).

Assessment of antigenic properties. As with library II, two assays were chosen for the assessment of the antigenic properties of the virus isolates: (i) the same type of ELISA used to characterize the subpools and (ii) the chimeric virus neutralization assay mediated by 2F5 (exemplified in Fig. 4). While not all viruses had outstanding scores in both assays (Table 2), we pursued 16 with at least one favorable score as well as manageable purification characteristics. In all but one case (chimeric virus 24-AN150-3), the individual isolates had significantly better scores (in at least one of the assays) than the parent pools (I, II, and III) from which they were originally derived.


Figure 4
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  		FIG. 4. ELISA (A) and neutralization (B) data representing the effects of immunoselection on the individual immunoselected chimeric viruses compared to those for their unselected parent pool. In this example, the starting set was pool II. The individual chimeras shown have been through two rounds of immunoselection. The presence of an N indicates that no competitive peptide was used. The subset numbers reflect the micromolar concentration of Ac-LELDKWASL-NH2 peptide used to compete with the chimeric viruses for binding to immobilized 2F5. In the panel A key, the final number is the virus clone number.


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  		TABLE 2. Summary of MAb 2F5 binding to and neutralization of the 16 immunoselected chimeric HRV14:HIV-1 ELDKWA viruses used to immunize guinea pigs and the unselected pools from which they were derived

Immunization of guinea pigs and characterization of immune responses. The 16 chosen chimeric viruses were each used to immunize three guinea pigs (which are not permissive for HRV replication) each. One of these chimeras, 14-C40-1, was used to immunize three extra guinea pigs (owing to promising results from the initial immunizations). Not knowing what might work best, immunization strategies were varied (Fig. 5), sometimes including one or two boosts with either of two KLH-conjugated ELDKWA-based peptides (with the sequences EQELLELDKWASLW [14-mer] and LELDKWASL [9-mer]).

The 51 serum samples generated were screened for their ability to neutralize HIV-1 pseudoviruses with envelopes from the following: MN laboratory isolate (clade B, X4), 92HT594 primary isolate (clade B, X4/R5), and QZ4589 primary isolate (clade B, R5). Based on the screening (data not shown), at least one serum sample elicited from each of six chimeras was chosen for more comprehensive testing; sera from a seventh chimera (44-B2.6-2) were chosen for their poor screening scores.

Figure 5 shows reciprocal neutralizing titers of serum samples (some IgG purified) resulting from immunizations with the seven chimeras (in some cases, with peptide boosts, from different stages of immunization, or from different animals). Figure S2 in the supplemental material shows representative neutralization curves. The sera were tested against seven pseudoviruses each (plus two more in the case of the most broadly neutralizing serum sample and its IgG-purified fraction), corresponding to HIV-1 isolates from diverse subtypes, sequences, and coreceptor usages. While significant animal-to-animal variation was observed, sera from all of the six favorably screened chimeras (i.e., not including 44-B2.6-2; some with peptide boosts) were able to neutralize at least one of the seven pseudoviruses tested. Three of the chimeras (14-B1.3-12, 14-C40-4, and 44-C40-4; some with peptide boosts) were able to elicit sera that neutralized at least four of the seven pseudoviruses tested. While many of the reciprocal neutralizing titers were modest, some corresponded to values in the hundreds.

The first serum listed (serum code 77; chimera 14-B1.3-12) neutralized all but two of the seven pseudoviruses tested. In the case of antiserum from virus 14-C40-1 (serum 63), there was no significant neutralizing activity by week 12 (i.e., no reciprocal titers of >20), but after a single boost at 13 weeks postimmunization with the KLH-conjugated 14-mer peptide (serum 63-P), the same guinea pig mounted neutralizing activity against five of seven pseudoviruses. The titers were better overall for serum 65-PP, from a guinea pig given the same immunization schedule as that used for 63-P except with an additional 14-mer peptide boost at week 9. Sera from two additional guinea pigs (66 and 67) were tested after immunization with the same 14-C40-1 virus that was followed by two 14-mer peptide boosts. In both cases, the number of pseudoviruses neutralized and the reciprocal neutralizing titers increased with the second boost: serum 66-P neutralized one pseudovirus after one peptide boost and two pseudoviruses after a second peptide boost (66-PP), and serum 67-P neutralized three pseudoviruses after one peptide boost and all seven pseudoviruses tested after the second peptide boost (67-PP). Given the breadth of neutralization by serum 67-PP, we tested its ability to neutralize two clade C pseudoviruses (98CN006 and 98IN022, previously shown to be susceptible to neutralization by the 4E10 MAb [directed against an immediately adjacent MPER epitope], but not the 2F5 MAb [11]). This serum was able to neutralize these pseudoviruses with reciprocal neutralizing titers of 100 and 95, respectively, despite the absence of the 4E10 epitope sequence. In most cases, increased breadth of neutralization was accompanied by an increased magnitude of pseudovirus neutralization.

Neutralizing potency elicited by another chimeric virus, 44-C40-4, was less sensitive to peptide boosting; however, the boost did increase the breadth of neutralization, resulting in the neutralization of two additional pseudoviruses. In this case, it could be significant that the shorter KLH-conjugated 9-mer peptide was used for the boosting and may not have had the equivalent boosting potential. Indeed, the 9-mer peptide is expected to be disordered (6, 13), in contrast to the 14-mer peptide (13), and it has been shown that longer peptides are better able to bind 2F5 (33), suggesting that a greater fraction of their structures are more likely to resemble appropriate structures for eliciting a 2F5-like response. Sera from the three isolates tested from pool II were seen to neutralize zero (44-B2.6-2, chosen for its lack of HIV-1 neutralization during screening) or three pseudoviruses each, while the only serum tested from a pool III-derived chimera, 24-AN150-3, neutralized two of the pseudoviruses.

Several controls were implemented to rule out the possibility that neutralization of the HIV-1 pseudoviruses by the guinea pig antisera was mediated by an inhibitory factor in the serum other than antibodies. Significantly, the most potent antiserum continued to neutralize HIV-1 pseudoviruses after IgG purification (67-PP-IgG) (Fig. 5), although the titers were reduced roughly threefold (possibly attributed to standard experimental error, considering that multiple procedures and assays were required for each comparison, and/or to the additional freeze-thaw of the serum required for IgG purification, and/or to removal of potentially neutralizing IgA antibodies [if present]). The persistence of the neutralizing activity, albeit less potent, indicates that antichimera IgG antibodies mediated at least some of the neutralization of diverse HIV-1 pseudoviruses.

To further address whether the observed HIV neutralization was specifically ELDKWA directed, ELISAs were done using immobilized 14-mer peptides to capture anti-ELDKWA antibodies. For the examples tested, ELDKWA-directed antibodies were seen with guinea pig sera exhibiting some of the more robust neutralizing responses (e.g., the 63 and 67 series) (Fig. 6A), whereas ELDKWA-directed antibodies were not seen with sera lacking neutralizing activity (e.g., the 147 and 149 series) (Fig. 6A). While this observation does not demonstrate that the ELDKWA-directed antibodies were, in fact, the antibodies that mediated the neutralization observed, it does correlate the presence of the ELDKWA-directed antibodies with the neutralizing ability of the sera against HIV-1 (Fig. 6B and 5).


Figure 6
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  		FIG. 6. ELISA and neutralization data for guinea pig antisera as a function of the immunization protocol. A. ELISA data show binding of sera to immobilized 14-mer peptide, Ac-EQELLELDKWASLW-NH2, for prebleeds, intermediate bleeds, and final bleeds from individual guinea pigs. B. A subset of the neutralization data of Fig. 5 is shown for the same samples used in the ELISAs (designated with the same symbols). The samples for which reciprocal neutralization titers were <20 are illustrated with values of slightly less than 20 for simplicity. Guinea pigs were immunized (as illustrated with arrowheads) at week 0 (V, 2.8 x 109 PFU chimeric virus), week 4 (V, 2.8 x 109 PFU chimeric virus), and week 9 (V, 2.8 x 109 PFU chimeric virus [for HRV14 and guinea pigs 63, 147, and 149] or half as much virus [1/2 V] plus half a dose of KLH-conjugated 14-mer peptide [1/2 P; 40 µg of the peptide moiety] for guinea pig 67), and week 13 (P, 80 µg of the 14-mer peptide moiety alone [or HRV14 alone]). Serum samples were collected at weeks –1, 12, and 16.

To further illustrate the specificity of the antisera, neutralization assays were done using a pseudovirus with the amphotropic murine leukemia virus ({alpha}-MLV) envelope. Although a number of sera elicited marginal neutralizing responses against the {alpha}-MLV pseudovirus (data not shown), there were no sera whose inhibition of {alpha}-MLV was consistently positive compared with HRV14 antiserum or pooled preimmune serum. Additionally, the prepared IgG purified fractions were unable to neutralize the {alpha}-MLV pseudovirus (see Fig. S2 in the supplemental material). Furthermore, preimmune sera and sera elicited by native rhinovirus (without the ELDKWA insert), and their IgG-purified fractions, did not neutralize any HIV-1 pseudoviruses.

The 14-mer peptide, which boosted the immune responses of chimera 14-C40-1, was unable to elicit neutralizing antibodies on its own. In an immunization scheme more rigorous than that used to boost 14-C40-1 (Fig. 5), the 14-mer alone did not elicit neutralizing antisera against any pseudovirus except 92TH024 (249-PPP-251-PPP), and this effect was eliminated after purification of the IgG antibodies. This observation suggests that the apparent neutralizing activity of serum 66-P versus 92TH024 could be attributable to the 14-mer peptide alone, as well, and not a result of IgG. However, it is worth noting that the 14-mer was unable to elicit neutralizing antisera against any pseudovirus when used more modestly to boost the 44-B2.6-2 chimera, which itself was ineffective in neutralizing any pseudoviruses (147 to 149). Together, these observations illustrate that the peptides alone did not account for the neutralizing activity seen with the 14-C40-1 samples. Likewise, no other ELDKWA-based peptides have been reported to elicit anti-HIV-1 neutralizing responses (35, 57).

Sera from many of the other chimeric viruses, in some cases boosted with ELDKWA-based peptides, did not elicit neutralizing responses against HIV-1 (Table 1 and data not shown), suggesting that only appropriately presented ELDKWA epitopes are able to elicit neutralizing responses.

Some of the sera have neutralizing activities with different specificity profiles than 2F5. An examination of 2F5 neutralizing activity (Fig. 5) reflects a range of greater than 3 orders of magnitude for the pseudoviruses tested. In contrast, the range of neutralizing activities was much narrower among many of the guinea pig sera (e.g., within a factor of severalfold). This greater consistency of response against the pseudoviruses could reflect that the guinea pig sera are polyclonal and at least some of the antibodies within are likely to have different neutralizing characteristics from 2F5 and/or recognize different chemical moieties or conformations of the ELDKWA region. In this regard, it could be significant that 2F5 most poorly neutralized the pseudoviruses with non-ELDKWA sequences (e.g., 92RW009 [ALDKWA], 92UG005 [QLDKWA], and 98CN006 and 98IN022 [both with ALDSWK]), whereas this effect was less pronounced with the polyclonal chimera antisera.


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DISCUSSION
 
In an effort to develop HIV vaccine components that can elicit broadly reactive neutralizing antibodies, we have displayed the conserved HIV-1 gp41 ELDKWA epitope in various ways on the relatively safe, immunogenic human rhinovirus. Three combinatorial libraries were produced, each containing the ELDKWA epitope grafted onto a surface loop of HRV via randomized, variable-length linker sequences. These libraries were expected to produce chimeric viruses presenting the HIV epitope in diverse conformations, some of which could resemble the cognate epitope that elicited the production of the broadly neutralizing MAb, 2F5. 2F5 was used to capture chimeric viruses presenting the ELDKWA epitope in antigenically relevant ways, in hopes that this would lead to chimeras with vaccine-caliber immunogenicity.

The first combinatorial library was ill fated, resulting in the production of viruses that had eliminated the foreign sequences (occasionally with parts of the adjoining HRV sequences). The second combinatorial library led to the production of live recombinant viruses with intact ELDKWA epitopes. However, none of the viruses tested from this library appeared to raise useful neutralizing antibodies in guinea pigs.

A third combinatorial library was described that included viruses capable of eliciting broadly neutralizing anti-HIV-1 antibodies, possibly benefited by the nonrandom occurrence of linker residues that promoted the formation of β-turns. Indeed, the 14-C40-1 chimeric virus, which produced the most cross-reactive neutralizing antibodies seen in this study (after a peptide boost), has the most extreme β-turn-forming tendency of the six most promising chimeras: 100% and 80% β-turn-promoting N and C linker randomized residues, respectively, versus an average of 60% and 47% β-turn-promoting N and C linker randomized residues, respectively, among all six chimeras with the most favorable screening characteristics. The β-turn-forming tendencies of the residues in this set of chimeras, perhaps initially required for viral fitness, may have fortuitously favored ELDKWA conformations resembling those of the 2F5 epitope as it appears in an immunogenic phase of the HIV life cycle. Some of the β-turn conformations may resemble that which was crystallographically determined for an ELDKWA-containing peptide when complexed with the 2F5 Fab fragment (64). Notably, studies with recombinant anti-HLA-DR gene products containing the ELDKWAS sequence have shown preferential binding to 2F5 when ELDKWAS is inserted into sites of putative β-turns, rather than helical conformations (32). Recent molecular dynamics simulations have shown that the ELDKWA sequence adopts both helical and β-turn conformations, with their relative abundances depending upon the context of the epitope (41).

Various 2F5-based immunoselection conditions (some containing competing peptide) were used to generate diverse virus pools. The virus pools that bound 2F5 most strongly, on average, were used as sources for finding the most promising chimeras. Ultimately, 15 chimeric viruses were chosen for immunogenicity studies in guinea pigs.

Using various immunization strategies, we identified chimeric viruses that elicited antibodies capable of neutralizing HIV-1 pseudoviruses of multiple clades and coreceptor usages. In one case, a chimeric virus/peptide immunization scheme produced sera able to neutralize pseudoviruses of clades B, A, A/E, D, and even C, which has been refractory to neutralization by 2F5. These results were obtained with sera derived from a guinea pig immunized with a chimeric ALDKWA derivative, 14-C40-1, and boosted with a conjugated 14-mer ELDKWA-based peptide. IgG purification of the broadly neutralizing 67-PP serum was used to demonstrate that the resultant HIV-1 neutralization was specifically antibody mediated. Furthermore, these neutralizing responses correlated with the presence of ELDKWA-directed antibodies, as seen in ELISAs with immobilized 14-mer ELDKWA peptide. While most of the sera described were depleted during this study, future studies will be conducted using IgG fractions, thus avoiding any serum non-IgG components that could influence neutralization.

Although ELISA-based recognition of the ELDKWA peptide by the ALDKWA-containing chimera antisera decreased with successive ELDKWA peptide boosts, both the potency and breadth of HIV neutralization improved (Fig. 6A and B). In contrast, the same peptide was unable to boost the responses of a poorly immunogenic chimeric virus or elicit IgG-based neutralization on its own. This could reflect an inability of the peptide to initiate potent B-cell responses. In contrast, virus-induced B-cell activation may allow peptides to boost the subset of the immune response that is, at least partially, ELDKWA directed. Such responses may result in antibodies with epitopes that either partially overlap the cognate ELDKWA epitope or perhaps recognize complementary conformations of sequences that contain either the A or E residues (i.e., the case above). Such antibodies could increase the breadth and potency of a polyclonal HIV-directed response. Some of the antibodies generated from the most promising chimera/peptide immunogens described may contain paratopes similar, but not identical, to that of 2F5, as demonstrated by their respective HIV neutralization profiles.

The apparent lack of 2F5-like neutralizing antibodies generated using recombinant immunogens (19, 24, 26, 33, 35, 38, 40, 42, 44-48, 50, 53, 54, 56, 57, 61, 63, 69, 93, 96) (see Table S1 in the supplemental material), or among HIV-infected individuals (10, 14, 15, 52, 61, 94), led Haynes et al. (31) to postulate that the 2F5 epitope acts as an autoantigen by mimicking the phospholipid cardiolipin. Given that "infectious autoantibodies to cardiolipin are rarely pathogenic and they are transient" (31), that passive immunization studies with this antibody have not led to observable autoimmune pathology (34, 87), and that recent papers have indicated no evidence for 2F5 reactivity with cardiolipin (55, 80, 90), mimicry may not be a significant problem with this epitope. In fact, reports have been mounting that counter the notion that such anti-MPER antibodies are extremely rare and, instead, provide a modicum of encouragement for the development of successful MPER-based vaccine immunogens (16, 29, 30, 68).

The work presented here as well as the work presented from many other labs shows the critical importance of the details of epitope presentation in eliciting relevant, effective antibodies. In the case of presenting the epitope in a safe, foreign context, this requirement is further complicated by the need to be compatible with the vector (demonstrated by the abundance of β-turn-promoting residues seen connecting the ELDKWA epitope to HRV) and still acquire conformations needed to elicit protective responses against the pathogen. It may be that some of the conserved residues surrounding the ELDKWA epitope in the context of HIV serve to enable the conformational changes necessary for fusion. These residues may not be necessary, however, for recombinant constructs to support conformations of the epitope that elicit protective immune responses.

This work describes the first recombinant MPER-based immunogen, and one of the only recombinant HIV-based immunogens, shown to elicit cross-reactive, albeit modest, neutralizing responses against HIV. While the immune responses elicited may need to be more potent, cross-reactive, or used in combination with additional HIV immunogens to be effective as vaccines, this system provides a foundation on which to build future constructs. In addition, variations on the presentation of ELDKWA within this system (and others) will hopefully begin to shed light on the connection between the structural and functional aspects of this immunogen. It is our hope that structural studies (using X-ray crystallography or cryo-electron microscopy) of the Fab fragment of 2F5 complexed with some of the chimeric viruses will contribute to an understanding of the observed differences in neutralization specificity.

It is not clear what attributes of the chimeras—and peptide boosts—are responsible for eliciting neutralizing antibodies against HIV. A more thorough analysis with a larger group of ELDKWA-presenting chimeras is in progress. Furthermore, since the mucosal immune response may be a critical factor in HIV protection, chimera-induced IgA production and activity will be examined. These studies may enable a better determination of virus characteristics or analytical assays that can help identify features of the chimeric virus immunogens that will have a positive impact on the immunological outcome.


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ACKNOWLEDGMENTS
 
We are grateful to Tom Ketas and John Moore for help with the PHA-stimulated PBMC neutralization assays used to assess the activity of the serum from the most promising chimeras from library II. We thank members of our laboratory, including Art Clark and Chhaya Dharia for assistance with the IgG purification and Allen Smith, Jianping Ding, Yu Hsiou, Deena Oren, Yulia Frenkel, and Kalyan Das for helpful discussions. We also thank Alan Schultz and Emily Carrow of the International AIDS Vaccine Initiative (IAVI) for encouragement and support.

This work was supported by grants to G.F.A. (NIH AI38221, AI45353, and AI071874, as well as financial support from IAVI) and to E.A. (NIH P01 GM066671).


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FOOTNOTES
 
* Corresponding author. Mailing address: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854-5638. Phone for G. F. Arnold: (732) 235-4343. Fax: 732-235-5788. E-mail: gfarnold{at}cabm.rutgers.edu. Phone for E. Arnold: (732) 235-5323. E-mail: arnold{at}cabm.rutgers.edu Back

{triangledown} Published ahead of print on 11 March 2009. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back

{ddagger} Present address: Abbott Laboratories, Bedford, MA. Back


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REFERENCES
 
    1
  1. Alfsen, A., and M. Bomsel. 2002. HIV-1 gp41 envelope residues 650-685 exposed on native virus act as a lectin to bind epithelial cell galactosyl ceramide. J. Biol. Chem. 277:25649-25659.[Abstract/Free Full Text]
    2. 2
  2. Alfsen, A., P. Iniguez, E. Bouguyon, and M. Bomsel. 2001. Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1. J. Immunol. 166:6257-6265.[Abstract/Free Full Text]
    3. 3
  3. Arnold, G. F., and E. Arnold. 1999. Using combinatorial libraries to develop vaccines. ASM News http://newsarchive.asm.org/sept99/feature2.asp.
    4. 4
  4. Arnold, G. F., D. A. Resnick, Y. Li, A. Zhang, A. D. Smith, S. C. Geisler, A. Jacobo-Molina, W.-M. Lee, R. G. Webster, and E. Arnold. 1994. Design and construction of rhinovirus chimeras incorporating immunogens from polio, influenza, and human immunodeficiency viruses. Virology 198:703-708.[CrossRef][Medline]
    5. 5
  5. Baba, T. W., V. Liska, R. Hofmann-Lehmann, J. Vlasak, W. Xu, S. Ayehunie, L. A. Cavacini, M. R. Posner, H. Katinger, G. Stiegler, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, Y. Lu, J. E. Wright, T. C. Chou, and R. M. Ruprecht. 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat. Med. 6:200-206.[CrossRef][Medline]
    6. 6
  6. Barbato, G., E. Bianchi, P. Ingallinella, W. H. Hurni, M. D. Miller, G. Ciliberto, R. Cortese, R. Bazzo, J. W. Shiver, and A. Pessi. 2003. Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion. J. Mol. Biol. 330:1101-1115.[CrossRef][Medline]
    7. 7
  7. Barin, F., G. Jourdain, S. Brunet, N. Ngo-Giang-Huong, S. Weerawatgoompa, W. Karnchanamayul, S. Ariyadej, R. Hansudewechakul, J. Achalapong, P. Yuthavisuthi, C. Ngampiyaskul, S. Bhakeecheep, C. Hemwutthiphan, and M. Lallemant. 2006. Revisiting the role of neutralizing antibodies in mother-to-child transmission of HIV-1. J. Infect. Dis. 193:1504-1511.[CrossRef][Medline]
    8. 8
  8. Belec, L., P. D. Ghys, H. Hocini, J. N. Nkengasong, J. Tranchot-Diallo, M. O. Diallo, V. Ettiegne-Traore, C. Maurice, P. Becquart, M. Matta, A. Si-Mohamed, N. Chomont, I. M. Coulibaly, S. Z. Wiktor, and M. D. Kazatchkine. 2001. Cervicovaginal secretory antibodies to human immunodeficiency virus type 1 (HIV-1) that block viral transcytosis through tight epithelial barriers in highly exposed HIV-1-seronegative African women. J. Infect. Dis. 184:1412-1422.[CrossRef][Medline]
    9. 9
  9. Beyrer, C., A. W. Artenstein, S. Rugpao, H. Stephens, T. C. VanCott, M. L. Robb, M. Rinkaew, D. L. Birx, C. Khamboonruang, P. A. Zimmerman, K. E. Nelson, C. Natpratan, et al. 1999. Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand. J. Infect. Dis. 179:59-67.[CrossRef][Medline]
    10. 10
  10. Binley, J. M., E. A. Lybarger, E. T. Crooks, M. S. Seaman, E. Gray, K. L. Davis, J. M. Decker, D. Wycuff, L. Harris, N. Hawkins, B. Wood, C. Nathe, D. Richman, G. D. Tomaras, F. Bibollet-Ruche, J. E. Robinson, L. Morris, G. M. Shaw, D. C. Montefiori, and J. R. Mascola. 2008. Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C. J. Virol. 82:11651-11668.[Abstract/Free Full Text]
    11. 11
  11. Binley, J. M., T. Wrin, B. Korber, M. B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C. J. Petropoulos, and D. R. Burton. 2004. Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J. Virol. 78:13232-13252.[Abstract/Free Full Text]
    12. 12
  12. Biron, Z., S. Khare, S. R. Quadt, Y. Hayek, F. Naider, and J. Anglister. 2005. The 2F5 epitope is helical in the HIV-1 entry inhibitor T-20. Biochemistry 44:13602-13611.[CrossRef][Medline]
    13. 13
  13. Biron, Z., S. Khare, A. O. Samson, Y. Hayek, F. Naider, and J. Anglister. 2002. A monomeric 310-helix is formed in water by a 13-residue peptide representing the neutralizing determinant of HIV-1 on gp41. Biochemistry 41:12687-12696.[CrossRef][Medline]
    14. 14
  14. Braibant, M., S. Brunet, D. Costagliola, C. Rouzioux, H. Agut, H. Katinger, B. Autran, and F. Barin. 2006. Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors: prevalence and association with neutralizing activity. AIDS 20:1923-1930.[Medline]
    15. 15
  15. Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M. Purtscher, G. Gruber, C. Tauer, F. Steindl, A. Jungbauer, and H. Katinger. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retrovir. 10:359-369.[Medline]
    16. 16
  16. Calarota, S., M. Jansson, M. Levi, K. Broliden, O. Libonatti, H. Wigzell, and B. Wahren. 1996. Immunodominant glycoprotein 41 epitope identified by seroreactivity in HIV type 1-infected individuals. AIDS Res. Hum. Retrovir. 12:705-713.[Medline]
    17. 17
  17. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273.[CrossRef][Medline]
    18. 18
  18. Chou, P. Y., and G. D. Fasman. 1974. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry 13:211-222.[CrossRef][Medline]
    19. 19
  19. Coeffier, E., J.-M. Clement, V. Cussac, N. Khodaei-Boorane, M. Jehanno, M. Rojas, A. Dridi, M. Latour, R. El Habib, F. Barre-Sinoussi, M. Hofnung, and C. Leclerc. 2000. Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein. Vaccine 19:684-693.[CrossRef][Medline]
    20. 20
  20. Conant, R. M., and V. V. Hamparian. 1968. Rhinoviruses: basis for a numbering system 1. HeLa cells for propagation and serologic procedures. J. Immunol. 100:107-113.[Abstract/Free Full Text]
    21. 21
  21. Conley, A. J., J. A. Kessler, II, L. J. Boots, J.-S. Tung, B. A. Arnold, P. M. Keller, A. R. Shaw, and E. A. Emini. 1994. Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody. Proc. Natl. Acad. Sci. USA 91:3348-3352.[Abstract/Free Full Text]
    22. 22
  22. Couch, R. B. 1996. Rhinoviruses, p. 713-734. In B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus (ed.), Fields virology, vol. 2, 3rd ed. Lippincott-Raven, Philadelphia, PA.
    23. 23
  23. Deeks, S. G., B. Schweighardt, T. Wrin, J. Galovich, R. Hoh, E. Sinclair, P. Hunt, J. M. McCune, J. N. Martin, C. J. Petropoulos, and F. M. Hecht. 2006. Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on the ability of HIV to completely evade neutralizing antibody responses. J. Virol. 80:6155-6164.[Abstract/Free Full Text]
    24. 24
  24. Devito, C., B. Zuber, U. Schroder, R. Benthin, K. Okuda, K. Broliden, B. Wahren, and J. Hinkula. 2004. Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity. J. Immunol. 173:7078-7089.[Abstract/Free Full Text]
    25. 25
  25. D'Souza, M. P., G. Milman, J. A. Bradac, D. McPhee, C. V. Hanson, R. M. Hendry, and C. Investigators. 1995. Neutralization of primary HIV-1 isolates by anti-envelope monoclonal antibodies. AIDS 9:867-874.[Medline]
    26. 26
  26. Eckhart, L., W. Rafflesberger, B. Ferko, A. Klima, M. Purtscher, H. Katinger, and F. Ruker. 1996. Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus. J. Gen. Virol. 77:2001-2008.[Abstract/Free Full Text]
    27. 27
  27. Fenyo, E. M., and P. Putkonen. 1996. Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections. Immunol. Lett. 51:95-99.[CrossRef][Medline]
    28. 28
  28. Fox, D. G., P. Balfe, C. P. Palmer, J. C. May, C. Arnold, and J. A. McKeating. 1997. Length polymorphism within the second variable region of the human immunodeficiency virus type 1 envelope glycoprotein affects accessibility of the receptor binding site. J. Virol. 71:759-765.[Abstract]
    29. 29
  29. Geffin, R. B., G. B. Scott, M. Melenwick, C. Hutto, S. Lai, L. J. Boots, P. M. McKenna, J. A. Kessler, II, and A. J. Conley. 1998. Association of antibody reactivity to ELDKWA, a glycoprotein 41 neutralization epitope, with disease progression in children perinatally infected with HIV type 1. AIDS Res. Hum. Retrovir. 14:579-590.[Medline]
    30. 30
  30. Gray, E. S., P. L. Moore, I. A. Choge, J. M. Decker, F. Bibollet-Ruche, H. Li, N. Leseka, F. Treurnicht, K. Mlisana, G. M. Shaw, S. S. Karim, C. Williamson, and L. Morris. 2007. Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection. J. Virol. 81:6187-6196.[Abstract/Free Full Text]
    31. 31
  31. Haynes, B. F., J. Fleming, E. W. St Clair, H. Katinger, G. Stiegler, R. Kunert, J. Robinson, R. M. Scearce, K. Plonk, H. F. Staats, T. L. Ortel, H. X. Liao, and S. M. Alam. 2005. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308:1906-1908.[Abstract/Free Full Text]
    32. 32
  32. Ho, J., K. S. MacDonald, and B. H. Barber. 2002. Construction of recombinant targeting immunogens incorporating an HIV-1 neutralizing epitope into sites of differine conformational constraint. Vaccine 20:1169-1180.[CrossRef][Medline]
    33. 33
  33. Ho, J., R. A. Uger, M. B. Zwick, M. A. Luscher, B. H. Barber, and K. S. MacDonald. 2005. Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response. Vaccine 23:1559-1573.[CrossRef][Medline]
    34. 34
  34. Joos, B., A. Trkola, H. Kuster, L. Aceto, M. Fischer, G. Stiegler, C. Armbruster, B. Vcelar, H. Katinger, and H. F. Gunthard. 2006. Long-term multiple-dose pharmacokinetics of human monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 envelope gp120 (MAb 2G12) and gp41 (MAbs 4E10 and 2F5). Antimicrob. Agents Chemother. 50:1773-1779.[Abstract/Free Full Text]
    35. 35
  35. Joyce, J. G., W. M. Hurni, M. J. Bogusky, V. M. Garsky, X. Liang, M. P. Citron, R. C. Danzeisen, M. D. Miller, J. W. Shiver, and P. M. Keller. 2002. Enchancement of β-helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. J. Biol. Chem. 277:45811-45820.[Abstract/Free Full Text]
    36. 36
  36. Karlsson Hedestam, G. B., R. A. Fouchier, S. Phogat, D. R. Burton, J. Sodroski, and R. T. Wyatt. 2008. The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus. Nat. Rev. Microbiol. 6:143-155.[CrossRef][Medline]
    37. 37
  37. Kaul, R., D. Trabattoni, J. J. Bwayo, D. Arienti, A. Zagliani, F. M. Mwangi, C. Kariuki, E. N. Ngugi, K. S. MacDonald, T. B. Ball, M. Clerici, and F. A. Plummer. 1999. HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers. AIDS 13:23-29.[CrossRef][Medline]
    38. 38
  38. Kim, M., Z. Qiao, J. Yu, D. Montefiori, and E. L. Reinherz. 2007. Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state. Vaccine 25:5102-5114.[CrossRef][Medline]
    39. 39
  39. Korber, B. T. M., C. Brander, B. F. Haynes, R. A. Koup, C. Kuiken, J. P. Moore, B. D. Walker, and D. I. Watkins (ed.). 2001. HIV Molecular Immunology 2001. Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, NM.
    40. 40
  40. Kusov, Y. Y., N. A. Zamjatina, V. F. Poleschuk, M. I. Michailov, G. Morace, J. Eberle, and V. Gauss-Muller. 2007. Immunogenicity of a chimeric hepatitis A virus (HAV) carrying the HIV gp41 epitope 2F5. Antivir. Res. 73:101-111.[CrossRef][Medline]
    41. 41
  41. Lapelosa, M., E. Gallicchio, G. F. Arnold, E. Arnold, and R. M. Levy. 2009. In silico vaccine design based on molecular simulations of rhinovirus chimeras presenting HIV-1 gp41 epitopes. J. Mol. Biol. 385:675-691.[CrossRef][Medline]
    42. 42
  42. Law, M., R. M. Cardoso, I. A. Wilson, and D. R. Burton. 2007. Antigenic and immunogenic study of membrane-proximal external region-grafted gp120 antigens by a DNA prime-protein boost immunization strategy. J. Virol. 81:4272-4285.[Abstract/Free Full Text]
    43. 43
  43. Lee, W.-M., S. S. Monroe, and R. R. Rueckert. 1993. Role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome. J. Virol. 67:2110-2122.[Abstract/Free Full Text]
    44. 44
  44. Lenz, O., M. T. Dittmar, A. Wagner, B. Ferko, K. Vorauer-Uhl, G. Stiegler, and W. Weissenhorn. 2005. Trimeric membrane-anchored gp41 inhibits HIV membrane fusion. J. Biol. Chem. 280:4095-4101.[Abstract/Free Full Text]
    45. 45
  45. Liang, X., S. Munshi, J. Shendure, G. MarkIii, M. E. Davies, D. C. Freed, D. C. Montefiori, and J. W. Shiver. 1999. Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein. Vaccine 17:2862-2872.[CrossRef][Medline]
    46. 46
  46. Liu, Z., Z. Wang, and Y.-H. Chen. 2005. Predefined spacers between epitopes on a recombinant epitope-peptide impacted epitope-specific antibody response. Immunol. Lett. 97:41-45.[CrossRef][Medline]
    47. 47
  47. Louis, J. M., C. A. Bewley, E. Gustchina, A. Aniana, and G. M. Clore. 2005. Characterization and HIV-1 fusion inhibitory properties of monoclonal Fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of HIV-1 gp41. J. Mol. Biol. 353:945-951.[CrossRef][Medline]
    48. 48
  48. Luo, M., F. Yuan, Y. Liu, S. Jiang, X. Song, P. Jiang, X. Yin, M. Ding, and H. Deng. 2006. Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment. Vaccine 24:435-442.[CrossRef][Medline]
    49. 49
  49. Manrique, A., P. Rusert, B. Joos, M. Fischer, H. Kuster, C. Leemann, B. Niederost, R. Weber, G. Stiegler, H. Katinger, H. F. Gunthard, and A. Trkola. 2007. In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10. J. Virol. 81:8793-8808.[Abstract/Free Full Text]
    50. 50
  50. Marusic, C., P. Rizza, L. Lattanzi, C. Mancini, M. Spada, F. Belardelli, E. Benvenuto, and I. Capone. 2001. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J. Virol. 75:8434-8439.[Abstract/Free Full Text]
    51. 51
  51. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb, H. Katinger, and D. L. Birx. 1999. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6D passive transfer of neutralizing antibodies. J. Virol. 73:4009-4018.[Abstract/Free Full Text]
    52. 52
  52. Mascola, J. R., M. K. Louder, T. C. Vancott, C. V. Sapan, J. S. Lambert, L. R. Muenz, B. Bunow, D. L. Birx, and M. L. Robb. 1997. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12. J. Virol. 71:7198-7206.[Abstract]
    53. 53
  53. Matoba, N., B. C. Geyer, J. Kilbourne, A. Alfsen, M. Bomsel, and T. S. Mor. 2006. Humoral immune responses by prime-boost heterologous route immunizations with CTB-MPR649-684, a mucosal subunit HIV/AIDS vaccine candidate. Vaccine 24:5047-5055.[CrossRef][Medline]
    54. 54
  54. Matoba, N., A. Magerus, B. C. Geyer, Y. Zhang, M. Muralidharan, A. Alfsen, C. J. Arntzen, M. Bomsel, and T. S. Mor. 2004. A mucosally targeted subunit vaccine candidate eliciting HIV-1 transcytosis-blocking Abs. Proc. Natl. Acad. Sci. USA 101:13584-13589.[Abstract/Free Full Text]
    55. 55
  55. Matyas, G. R., Z. Beck, N. Karasavvas, and C. R. Alving. 2009. Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1. Biochim. Biophys. Acta 1788:660-665.[Medline]
    56. 56
  56. McGaughey, G. B., G. Barbato, E. Bianchi, R. M. Freidinger, V. M. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M. D. Miller, A. Pessi, J. W. Shiver, and M. J. Bogusky. 2004. Progress towards the development of a HIV-1 gp41-directed vaccine. Curr. HIV Res. 2:193-204.[CrossRef][Medline]
    57. 57
  57. McGaughey, G. B., M. Citron, R. C. Danzeisen, R. M. Freidinger, V. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M. Miller, J. Shiver, and M. J. Bogusky. 2003. HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb. Biochemistry 42:3214-3223.[CrossRef][Medline]
    58. 58
  58. Montefiori, D., Q. Sattentau, J. Flores, J. Esparza, and J. Mascola. 2007. Antibody-based HIV-1 vaccines: recent developments and future directions. PLoS Med. 4:e348.[CrossRef][Medline]
    59. 59
  59. Montero, M., N. E. van Houten, X. Wang, and J. K. Scott. 2008. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design. Microbiol. Mol. Biol. Rev. 72:54-84.[Abstract/Free Full Text]
    60. 60
  60. Munoz-Barroso, I., K. Salzwedel, E. Hunter, and R. Blumenthal. 1999. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type I envelope glycoprotein-mediated membrane fusion. J. Virol. 73:6089-6092.[Abstract/Free Full Text]
    61. 61
  61. Muster, T., R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger. 1994. Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. J. Virol. 68:4031-4034.[Abstract/Free Full Text]
    62. 62
  62. Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647.[Abstract/Free Full Text]
    63. 63
  63. Ni, J., R. Powell, I. V. Baskakov, A. DeVico, G. K. Lewis, and L.-X. Wang. 2004. Synthesis, conformation, and immunogenicity of monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178. Bioorg. Med. Chem. 12:3141-3148.[CrossRef][Medline]
    64. 64
  64. Ofek, G., M. Tang, A. Sambor, H. Katinger, J. R. Mascola, R. Wyatt, and P. D. Kwong. 2004. Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J. Virol. 78:10724-10737.[Abstract/Free Full Text]
    65. 65
  65. Pantaleo, G., S. Menzo, M. Vaccarezza, C. Graziosi, O. Cohen, J. F. Demarest, D. Montefiori, J. M. Orenstein, C. Fox, L. K. Schrager, J. B. Margolick, S. Buchbinder, J. Giorgi, and A. Fauci. 1995. Studies in subjects with long-term nonprogressive human immunodeficiency virus infection. N. Engl. J. Med. 332:209-216.[Abstract/Free Full Text]
    66. 66
  66. Parren, P. W., P. A. Marx, A. J. Hessell, A. Luckay, J. Harouse, C. Cheng-Mayer, J. P. Moore, and D. R. Burton. 2001. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J. Virol. 75:8340-8347.[Abstract/Free Full Text]
    67. 67
  67. Parren, P. W. H., I. D. R. Buron, and Q. Sattentau. 1997. HIV-1 antibody-debris or virion? Nat. Med. 3:366-367.[Medline]
    68. 68
  68. Penn-Nicholson, A., D. P. Han, S. J. Kim, H. Park, R. Ansari, D. C. Montefiori, and M. W. Cho. 2008. Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope. Virology 372:442-456.[CrossRef][Medline]
    69. 69
  69. Phogat, S., K. Svehla, M. Tang, A. Spadaccini, J. Muller, J. Mascola, I. Berkower, and R. Wyatt. 2008. Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles. Virology 373:72-84.[CrossRef][Medline]
    70. 70
  70. Piacentini, L., C. Fenizia, V. Naddeo, and M. Clerici. 2008. Not just sheer luck! Immune correlates of protection against HIV-1 infection. Vaccine 26:3002-3007.[CrossRef][Medline]
    71. 71
  71. Pinter, A., W. J. Honnen, Y. He, M. K. Gorny, S. Zolla-Pazner, and S. C. Kayman. 2004. The V1/V2 domain of gp120 is a global regulator of the sensitivity of primary human immunodeficiency virus type 1 isolates to neutralization by antibodies commonly induced upon infection. J. Virol. 78:5205-5215.[Abstract/Free Full Text]
    72. 72
  72. Poignard, P., E. O. Saphire, P. W. Parren, and D. R. Burton. 2001. gp120: biologic aspects of structural features. Annu. Rev. Immunol. 19:253-274.[CrossRef][Medline]
    73. 73
  73. Purtscher, M., A. Trkola, G. Gruber, A. Buchacher, R. Predl, F. Steindl, C. Tauer, R. Berger, N. Barrett, A. Jungbauer, and H. Katinger. 1994. A broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1. AIDS Res. Hum. Retrovir. 10:1651-1658.[Medline]
    74. 74
  74. Quinnan, G. V., Jr., X.-F. Yu, M. G. Lewis, P. F. Zhang, G. Sutter, P. Silvera, M. Dong, A. Choudhary, P. T. N. Sarkis, P. Bouma, Z. Zhang, D. C. Montefiori, T. C. VanCott, and C. C. Broder. 2005. Protection of rhesus monkeys against infection with minimally pathogenic simian-human immunodeficiency virus: correlations with neutralizing antibodies and cytotoxic T cells. J. Virol. 79:3358-3369.[Abstract/Free Full Text]
    75. 75
  75. Resnick, D. A., A. D. Smith, S. C. Geisler, A. Zhang, E. Arnold, and G. F. Arnold. 1995. Chimeras from a human rhinovirus 14-human immunodeficiency virus type 1 (HIV-1) V3 loop seroprevalence library induce neutralizing responses against HIV-1. J. Virol. 69:2406-2411.[Abstract]
    76. 76
  76. Richman, D. D., T. Wrin, S. J. Little, and C. J. Petropoulos. 2003. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc. Natl. Acad. Sci. USA 100:4144-4149.[Abstract/Free Full Text]
    77. 77
  77. Rossmann, M. G., E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend. 1985. Structure of a human common cold virus and functional relationships to other picornaviruses. Nature 317:145-153.[CrossRef][Medline]
    78. 78
  78. Salzwedel, K., J. T. West, and E. Hunter. 1999. A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity. J. Virol. 73:2469-2480.[Abstract/Free Full Text]
    79. 79
  79. Scarlatti, G., J. Albert, P. Rossi, V. Hodara, P. Biraghi, L. Muggiasca, and E. M. Fenyo. 1993. Mother-to-child transmission of human immunodeficiency virus type 1: correlation with neutralizing antibodies against primary isolates. J. Infect. Dis. 168:207-210.[Medline]
    80. 80
  80. Scherer, E. M. A., M. B. A. Zwick, L. A. Teyton, and D. R. A. B. Burton. 2007. Difficulties in eliciting broadly neutralizing anti-HIV antibodies are not explained by cardiolipin autoreactivity. AIDS 21:2131-2139.[Medline]
    81. 81
  81. Sherry, B., and R. R. Rueckert. 1985. Evidence for at least two dominant neutralization antigens on human rhinovirus 14. J. Virol. 53:137-143.[Abstract/Free Full Text]
    82. 82
  82. Smith, A. D., S. C. Geisler, A. A. Chen, D. A. Resnick, B. M. Roy, P. J. Lewi, E. Arnold, and G. F. Arnold. 1998. Human rhinovirus type 14:human immunodeficiency virus type 1 (HIV-1) V3 loop chimeras from a combinatorial library induce potent neutralizing antibody responses against HIV-1. J. Virol. 72:651-659.[Abstract/Free Full Text]
    83. 83
  83. Smith, A. D., D. A. Resnick, A. Zhang, S. C. Geisler, E. Arnold, and G. F. Arnold. 1994. Use of random systematic mutagenesis to generate viable human rhinovirus 14 chimeras displaying HIV-1 V3 loop sequences. J. Virol. 68:575-579.[Abstract/Free Full Text]
    84. 84
  84. Smith, D. M., M. C. Strain, S. D. Frost, S. K. Pillai, J. K. Wong, T. Wrin, Y. Liu, C. J. Petropolous, E. S. Daar, S. J. Little, and D. D. Richman. 2006. Lack of neutralizing antibody response to HIV-1 predisposes to superinfection. Virology 355:1-5.[CrossRef][Medline]
    85. 85
  85. Stiegler, G., R. Kunert, M. Purtscher, S. Wolbank, R. Voglauer, F. Steindl, and H. Katinger. 2001. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res. Hum. Retrovir. 17:1757-1765.[CrossRef][Medline]
    86. 86
  86. Tan, K., J. Liu, J. Wang, S. Shen, and M. Lu. 1997. Atomic structure of a thermostable subdomain of HIV-1 gp41. Proc. Natl. Acad. Sci. USA 94:12303-12308.[Abstract/Free Full Text]
    87. 87
  87. Trkola, A., H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. A. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard. 2005. Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies. Nat. Med. 11:615-622.[CrossRef][Medline]
    88. 88
  88. Trkola, A., A. B. Pomales, H. Yuan, B. Korber, P. J. Maddon, G. P. Allaway, H. Katinger, C. F. Barbas III, D. R. Burton, D. D. Ho, and J. P. Moore. 1995. Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J. Virol. 69:6609-6617.[Abstract]
    89. 89
  89. Ugen, K. E., J. J. Goedert, J. Boyer, Y. Refaeli, I. Frank, W. V. Williams, A. Willoughby, S. Landesman, H. Mendez, A. Rubinstein, T. Kieber-Emmons, and D. B. Weiner. 1992. Vertical transmission of human immunodeficiency virus (HIV) infection reactivity of maternal sera with glycoprotein 120 and 41 peptides from HIV type 1. J. Clin. Investig. 89:1923-1930.[Medline]
    90. 90
  90. Vcelar, B. A., G. A. Stiegler, H. M. B. Wolf, W. E. Muntean, B. E. Leschnik, S. F. Mehandru, M. F. Markowitz, C. C. Armbruster, R. D. Kunert, M. M. B. Eibl, and H. A. D. Katinger. 2007. Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data. AIDS 21:2161-2170.[CrossRef][Medline]
    91. 91
  91. Wei, X., J. M. Decker, S. Wang, H. Hul, J. C. Kappes, X. Wu, J. F. Salazar-Gonzalez, M. G. Salazar, J. M. Kilby, M. Saag, S. N. L. Komarova, M. A. Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003. Antibody neutralization and escape by HIV-1. Nature 422:307-312.[CrossRef][Medline]
    92. 92
  92. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387:426-430.[CrossRef][Medline]
    93. 93
  93. Ye, L., Y. Sun, J. Lin, Z. Bu, Q. Wu, S. Jiang, D. A. Steinhauer, R. W. Compans, and C. Yang. 2006. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein. Virology 352:74-85.[CrossRef][Medline]
    94. 94
  94. Yuste, E., H. B. Sanford, J. Carmody, J. Bixby, S. Little, M. B. Zwick, T. Greenough, D. R. Burton, D. D. Richman, R. C. Desrosiers, and W. E. Johnson. 2006. Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma. J. Virol. 80:3030-3041.[Abstract/Free Full Text]
    95. 95
  95. Zhang, A., R. G. Nanni, T. Li, G. F. Arnold, D. A. Oren, A. Jacobo-Molina, R. L. Williams, G. Kamer, D. A. Rubenstein, Y. Li, E. Rozhon, S. Cox, P. Buontempo, J. O'Connell, J. Schwartz, G. Miller, B. Bauer, R. Versace, P. Pinto, A. Ganguly, V. Girijavallabhan, and E. Arnold. 1993. Structure determination of antiviral compound SCH 38057 complexed with human rhinovirus 14. J. Mol. Biol. 230:857-867.[CrossRef][Medline]
    96. 96
  96. Zhang, H., Y. Huang, R. Fayad, G. T. Spear, and L. Qiao. 2004. Induction of mucosal and systemic neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) by oral immunization with bovine papillomavirus-HIV-1 gp41 chimeric virus-like particles. J. Virol. 78:8342-8348.[Abstract/Free Full Text]
    97. 97
  97. Zwick, M. B. 2005. The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring. AIDS 19:1725-1737.[Medline]
    98. 98
  98. Zwick, M. B., R. Jensen, S. Church, M. Wang, G. Stiegler, R. Kunert, H. Katinger, and D. R. Burton. 2005. Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1. J. Virol. 79:1252-1261.[Abstract/Free Full Text]

Journal of Virology, May 2009, p. 5087-5100, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.00184-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bryson, S., Julien, J.-P., Hynes, R. C., Pai, E. F. (2009). Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications. J. Virol. 83: 11862-11875 [Abstract] [Full Text]  

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