mAb 2F5 Research Articles

The Recombinant Immunogen with High‐Density Epitopes of ELDKWA and ELDEWA Induced Antibodies Recognizing Both Epitopes on HIV‐1 gp41

The high mutation rate of HIV-1 (human immunodeficiency virus-1) is a major obstacle to developing an effective vaccine. The mutation of ELDKWA-(aa669-674) to ELDEWA-epitope on HIV-1 gp41 caused the immune escape from neutralization by potent anti-HIV-1 human monoclonal antibody (mAb) 2F5. In this study, we suggested and evaluated a multi-epitope vaccine as a new strategy to develop HIV-1 vaccines. A glutathione S-transferase (GST) fusion protein (GST-K8E8) containing 8 copies of ELDKWA-and mutated ELDEWA-epitopes was constructed and used to immunize mice or rabbits. Analysis of the antisera (rAS3) induced by GST-K8E8 suggested that multi-epitope vaccine immunogen could raise antibodies in mice and rabbits against either the original ELDKWA-epitope or the mutated ELDEWA-epitope that resulted in immune escape. Briefly, ELDKWA-epitope-specific antibodies, directly purified from rAS3 by ELDKWA-epitope-peptide affinity chromatography, recognized either original gp41 protein (ELDKWA, rgp41K) or mutated gp41 (ELDEWA, rgp41E) in immunoblotting assay; in contrast, the existing ELDKWA-epitope antibodies recognized only rgp41K but not rgp41E, which were purified by ELDKWA-epitope-peptide affinity chromatography from rAS3 that were firstly completely pre-absorbed by ELDEWA-epitope-peptide affinity beads. And the same results were also observed when detecting the ELDEWA-epitope-specific antibodies in rAS3 by a means similar to the above. All the data presented here demonstrated that a high density multi-epitope vaccine could be an interesting strategy against HIV-1 mutation.

Virus Isolates during Acute and Chronic Human Immunodeficiency Virus Type 1 Infection Show Distinct Patterns of Sensitivity to Entry Inhibitors

We studied the effect of entry inhibitors on 58 virus isolates derived during acute and chronic infection to validate these inhibitors in vitro and to probe whether viruses at early and chronic disease stages exhibit general differences in the interaction with entry receptors. We included members of all types of inhibitors currently identified: (i) agents that block gp120 binding to CD4 (CD4-IgG2 and monoclonal antibody [MAb] IgG1b12), (ii) compounds that block the interaction with CCR5 (the chemokine RANTES/CCL5, the small-molecule inhibitor AD101, and the anti-CCR5 antibody PRO 140), (iii) the fusion inhibitor enfuvirtide (T-20), and (iv) neutralizing antibodies directed against gp120 (MAb 2G12) and gp41 (MAbs 2F5 and 4E10). No differences between viruses from acute and chronic infections in the susceptibility to inhibitors targeting the CD4 binding site, CCR5, or fusion or to MAb 2G12 were apparent, rendering treatment with entry inhibitors feasible across disease stages. The notable exceptions were antibodies 2F5 and 4E10, which were more potent in inhibiting viruses from acute infection (P = 0.0088 and 0.0005, respectively), although epitopes of these MAbs were equally well preserved in both groups. Activities of these MAbs correlated significantly with each other, suggesting that common features of the viral envelope modulate their potencies.

The neutralizing epitope ELDKWA on HIV-1 gp41: Genetic variability and antigenicity

Viral evasion through mutations of neutralizing epitopes is a major challenge to AIDS vaccine development and clinical therapy. In this study, we analyzed the genetic variability of principal neutralizing epitope ELDKWA among 5393 HIV isolates in the HIV sequence database ( http://www.hiv.lanl.gov). It has been found that six out of seven frequent variants were able to cause the invalidation of famous neutralizing MAb 2F5. Moreover, escape mutations K665S and K665E, often observed in C-subtype and O-group strains, respectively, were reported more frequently in recent years. Furthermore, we studied the antigenicity of these variants. Polyclonal antibodies induced by a candidate multi-epitope-vaccine were able to react with the recombinant gp41, respectively, carrying these variants, indicating that “epitope cocktail” strategy is necessary and helpful in vaccine design.

The C108g Epitope in the V2 Domain of gp120 Functions as a Potent Neutralization Target When Introduced into Envelope Proteins Derived from Human Immunodeficiency Virus Type 1 Primary Isolates

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.

Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies

The target for neutralizing antibodies against human immunodeficiency virus (HIV) is the trimeric Env protein on the native virion. Conserved neutralizing epitopes of receptor binding sites are located in the recessed core of the Env protein, partially masked by glycosylations and variable loops. In this study, we have investigated the effects of modifications of the HIV Env protein by glycosylation site mutations, deletions of variable loops, or combinations of both types of mutations on their protein functions and reactivities with neutralizing antibodies. Modified Env proteins were expressed in insect or mammalian cells, and their reactivity with epitope-specific broadly neutralizing monoclonal antibodies (Mabs) was determined by flow cytometry. A unique mutant designated 3G with mutations in three glycosylation motifs within the V3/C3 domains surrounding the CD4 binding site showed higher levels of binding to most broadly neutralizing Mabs (b12 and 2F5) in both insect and mammalian expression systems. Mutants with a deletion of both V1 and V2 loop domains or with a unique combination of both types of mutations also bound to most neutralizing Mabs at higher levels compared to the wild-type control. Most mutants maintained the ability to bind CD4 and to induce syncytium formation at similar or higher levels as compared to that of the wild-type Env protein, except for a mutant with a combination of variable loop deletions and deglycosylation mutations. Our study suggests that modified HIV Env proteins with reduced glycosylation in domains surrounding the CD4 binding site or variable loop-deleted mutants expose important neutralizing epitopes at higher levels than wild type and may provide novel vaccine immunogens.

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies.

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (</=7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV(+) plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade.

Predefined spacers between epitopes on a recombinant epitope-peptide impacted epitope-specific antibody response

We have developed a widely applicable method to construct epitope-peptide gene for epitope-vaccine strategy recently. In this study, we wanted to know whether the predefined spacers between epitopes on a recombinant epitope-peptide impacted the production of epitope-specific antibodies. The neutralizing epitope ELDKWA on the C-domain of HIV-1 gp41 was defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. We constructed three recombinant ELDKWA-epitope-peptides with different spacers between epitopes. The recombinant epitope-peptide GST-K 8, GST-S 8 and GST-R 8 were bearing eight copies of ELDKWA-epitope with amino acid spacer GS, GSGGGGS and RS, respectively. GST-K 8 and GST-S 8 could induce high titer of ELDKWA-epitope-specific antibodies, much better than GST-R 8. Besides, both antibodies could recognize the recombinant soluble gp41 and the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that the spacer GSGGGGS and GS were feasible in constructing a recombinant epitope-vaccine.

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.

A major problem hampering the development of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) is the resistance of many primary viral isolates to antibody-mediated neutralization. To identify factors responsible for this resistance, determinants of the large differences in neutralization sensitivities of HIV-1 pseudotyped with Env proteins derived from two prototypic clade B primary isolates were mapped. SF162 Env pseudotypes were neutralized very potently by a panel of sera from HIV-infected individuals, while JR-FL Env pseudotypes were neutralized by only a small fraction of these sera. This differential sensitivity to neutralization was also observed for a number of monoclonal antibodies (MAbs) directed against sites in the V2, V3, and CD4 binding domains, despite often similar binding affinities of these MAbs towards the two soluble rgp120s. The neutralization phenotypes were switched for chimeric Envs in which the V1/V2 domains of these two sequences were exchanged, indicating that the V1/V2 region regulated the overall neutralization sensitivity of these Envs. These results suggested that the inherent neutralization resistance of JR-FL, and presumably of related primary isolates, is to a great extent mediated by gp120 V1/V2 domain structure rather than by sequence variations at the target sites. Three MAbs (immunoglobulin G-b12, 2G12, and 2F5) previously reported to possess broad neutralizing activity for primary HIV-1 isolates neutralized JR-FL virus at least as well as SF162 virus and were not significantly affected by the V1/V2 domain exchanges. The rare antibodies capable of neutralizing a broad range of primary isolates thus appeared to be targeted to exceptional epitopes that are not sensitive to V1/V2 domain regulation of neutralization sensitivity.

Progress towards the development of a HIV-1 gp41-directed vaccine.

The HIV-1 gp41 envelope glycoprotein mediates fusion of the viral and cellular membranes. The core of the gp41 ectodomain undergoes a receptor-triggered conformational transition forming a trimeric, alpha-helical coiled-coil structure. This trimer-of-hairpins species facilitates insertion of the viral envelope protein into the host cell membrane promoting viral entry. The prefusogenic conformation of gp41 is capable of stimulating a neutralizing antibody immune response and is therefore an attractive therapeutic target. Several broadly neutralizing HIV-1 monoclonal antibodies which bind to gp41 have been characterized and include 4E10, Z13 and 2F5. A conserved segment of gp41 (residues 661-684) has been identified as the epitope for the HIV-1 neutralizing antibody 2F5 (MAb 2F5). MAb 2F5 has attracted considerable attention because of the highly conserved recognition epitope and the ability to neutralize both laboratory-adapted and primary viral isolates. Antibodies which recognize the immunodominant regions of gp41 may provide protection against HIV infection if elicited at appropriate concentrations. Here we review the rational design, structure-activity relationships and conformational features of both linear and constrained peptide immunogens incorporating variants of both the 2F5 epitope and the gp41 ectodomain. This review describes a rational design approach combining structural characterization with traditional SAR to optimize MAb 2F5 antibody affinities of gp41-based peptide immunogens. The immunogens are shown to stimulate a high titer, peptide-specific immune response; however, the resulting antisera were incapable of viral neutralization. The implication of these findings with regard to structural and immunological considerations is discussed.

Cellular immunity elicited by human immunodeficiency virus type 1/ simian immunodeficiency virus DNA vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies.

High levels of infused anti-human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibodies (MAbs) can completely protect macaque monkeys against mucosal chimeric simian-human immunodeficiency virus (SHIV) infection. Antibody levels below the protective threshold do not prevent infection but can substantially reduce plasma viremia. To assess if HIV-1/SIV-specific cellular immunity could combine with antibodies to produce sterile protection, we studied the effect of a suboptimal infusion of anti-HIV-1 neutralizing antibodies in macaques with active cellular immunity induced by interleukin-2 (IL-2)-adjuvanted DNA immunization. Twenty female macaques were divided into four groups: (i). DNA immunization plus irrelevant antibody, (ii). DNA immunization plus infusion of neutralizing MAbs 2F5 and 2G12, (iii). sham DNA plus 2F5 and 2G12, and (iv). sham DNA plus irrelevant antibody. DNA-immunized monkeys developed CD4 and CD8 T-cell responses as measured by epitope-specific tetramer staining and by pooled peptide ELISPOT assays for gamma interferon-secreting cells. After vaginal challenge, DNA-immunized animals that received irrelevant antibody became SHIV infected but displayed lower plasma viremia than control animals. Complete protection against SHIV challenge occurred in three animals that received sham DNA plus MAbs 2F5 and 2G12 and in two animals that received the DNA vaccine plus MAbs 2F5 and 2G12. Thus, although DNA immunization produced robust HIV-specific T-cell responses, we were unable to demonstrate that these responses contributed to the sterile protection mediated by passive infusion of neutralizing antibodies. These data suggest that although effector T cells can limit viral replication, they are not able to assist humoral immunity to prevent the establishment of initial infection.

Structural Analysis of the Epitope of the Anti-HIV Antibody 2F5 Sheds Light into Its Mechanism of Neutralization and HIV Fusion

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I β turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb.

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.

Immunogenic Properties of Peptides Mimicking a HIV-1 gp41 Epitope Recognized by Virus-Neutralizing Antibodies 2F5

Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (mAb) 2F5. Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages. Antibodies to gp41 were detected in the sera of immunized animals. The virus-neutralizing activity of the sera was examined.

ELDKWA-epitope-specific monoclonal antibodies inhibit HIV env-mediated syncytium formation

Recent studies demonstrated that the N- and C-domains of HIV-1 gp41 are involved in virus-mediated membrane fusion resulting in HIV-entry into target cells. Synthetic N- and C-domain peptides, such as DP107 and DP178, potently inhibit membrane fusion induced by both laboratory-adapted strains and primary isolates of HIV-1. Monoclonal antibody (mAb) 2F5 recognizing ELDKWA-epitope on the gp41 C-domain has shown broad neutralizing activity to many HIV stains even primary isolates. To test the neutralizing mechanism of the ELDKWA-epitope-specific antibody, mAbs with predefined ELDKWA-epitope-specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. All of five mAbs were identified to recognize ELDKWA-epitope on recombinant soluble gp41 in ELISA-assay. In flow cytometry analysis, four out of five mAbs could bind to HIV-Env+ CHO-WT cells and such binding could be inhibited by ELDKWA-epitope peptide, which suggests that these mAbs could recognize native envelope protein expressed on cell membranes. Interestingly, two out of five mAbs could inhibit membrane fusion between the CXCR4- and CD4-expressing 3T3.T4.CXCR4 cells and HIV-Env+ CHO-WT cells in a dose-dependent manner, consistent with their potent binding capability to HIV-Env+ CHO-WT cells. Parren et al. (1998) suggested that neutralization of HIV-1 by antibody may be determined primarily by occupancy of sites and spatial obstruction which blocks virus entry. Based on this theory, a model was suggested to explain the neutralizing activities of mAbs against the epitope ELDKWA.

Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64.

The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pH-dependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.

A predefined epitope-specific monoclonal antibody recognizes ELDEWA-epitope just presenting on gp41 of HIV-1 O clade

Diverse variation of HIV-1 is a grave challenge for prevention of viral infection and immunotherapy. Monoclonal antibody (mAb) 2F5 recognizing an epitope EL DK WA (aa669–674) on HIV-1 envelope protein gp41 showed broad neutralizing activity against a lot of HIV-1 strains including primary isolates. However, viral mutation from EL DK WA to ELD E̱WA resulted in viral evasion from neutralization by mAb 2F5. Using ELD E̱WA-epitope–peptide (C-GFLDEWAGELDEWA) conjugated with carrier protein keyhole limpet hemocyanin (KLH), a mAb 14D9 (IgGl) was prepared and identified as the mAb with predefined ELDEWA-epitope specificity. The mAb 14D9 recognized the ELD E̱WA epitope, but not other three epitopes (EL DK WA, EL ṈKWA and EL E̱KWA). In comparison, mAb 2F5 could recognize only EL DK WA, but not three neutralization-resistant epitopes (ELD E̱WA, EL ṈKWA and EL E̱KWA). Interestingly, we searched several authoritative HIV sequence databases ( http://hiv-web.lanl.gov) and found out that nearly all the viral isolates bearing the ELD E̱WA epitope belong to the O clade, the only exceptional viral isolate bearing the epitope ELD E̱WA has been demonstrated to be an intergroup M/O recombinant, which suggests that the ELD E̱WA-epitope on gp41 represents a specific epitope-marker of HIV-1 O clade. To confirm whether the mAb 14D9 recognizes gp41 of HIV-1 O clade, the rsgp41 IIIB, bearing ELD ḴWA-epitope was site-directed-mutated to the rsgp41 bearing ELD E̱WA-epitope. The mAb 14D9 could bind to rsgp41 bearing ELD E̱WA-epitope in immunoblotting analysis, did not bind to rsgp41 bearing ELD ḴWA-epitope. These experimental results suggest that the mAb 14D9 with predefined ELD E̱WA-epitope specificity may be applied to HIV-1 O clade identification.

Structure-affinity relationships in the gp41 ELDKWA epitope for the HIV-1 neutralizing monoclonal antibody 2F5: effects of side-chain and backbone modifications and conformational constraints.

The human monoclonal antibody, mAb 2F5, has broad HIV-1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high-affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme-linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end-capped, linear nonapeptide, Ac-LELDKWASL-amide, was sufficient to attain maximal affinity within the set of native gp41-sequence peptides assayed. Scanning single-residue alanine and d-residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high-affinity binding of the linear nonapeptide. Further studies of side-chain and backbone-modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu(3) and Trp(6) residues. The nine-residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15-residue, disulfide-bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C-peptide helix immediately adjacent to the N-terminal end of the ELDKWA epitope were examined through the synthesis of helix-initiated analogs. Circular dichroism (CD) studies indicated that the alpha-helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV-neutralizing actions of 2F5.

Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies.

Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.

Cross-Neutralizing Human Monoclonal Anti-HIV-1 Antibody 2F5: Preparation and Crystallographic Analysis of the Free and Epitope-Complexed Forms of its F ab Fragment

The human monoclonal antibody 2F5 is a potent neutralizer of most clades of HIV-1 and possesses protective effects against viral transmission. It recognizes the linear epitope ELDKWAS of the viral envelope protein gp41. As structural information about epitope recognition may help to develop an HIV-1 vaccine we initiated crystallographic analyses of mAb 2F5 and its epitope complex. We now report the preparation of the corresponding Fab fragments, complexation with the epitope peptide, and crystallization of free mAb 2F5 Fab as well as the peptide complex. Both crystal forms are well suited for high-resolution structural analysis.

Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge.

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.