Production of a novel antigen by conjugation of HIV-1 to Brucella abortus: studies of immunogenicity, isotype analysis, T-cell dependency, and syncytia inhibition.

In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.     

Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis.

The binding region for human immunodeficiency virus (HIV) and epitopes for a panel of HIV-blocking anti-CD4 monoclonal antibodies of the CD4 molecule were defined by using in vitro site-directed mutagenesis. Codons for two amino acid residues (Ser-Arg) were inserted at selected positions within the region encoding the first and second immunoglobulin-like domains of CD4. A vaccinia virus-based expression system was used to produce soluble full-length extracellular CD4 fragments containing the insertions. The mutant proteins were tested for direct binding to soluble gp120 (the CD4-binding subunit of the viral envelope glycoprotein) and to a series of HIV-blocking anti-CD4 monoclonal antibodies. Impaired gp120 binding activity resulted from insertions after amino acid residues 31, 44, 48, 52, 55, and 57 in the first immunoglobulin-like domain. The epitopes for two HIV-blocking monoclonal antibodies, OKT4A and OKT4D, were also mapped in the gp120-binding region in the first domain. Insertions after amino acid residues 21 and 91 in the first domain had no effect on gp120 binding but impaired the binding of OKT4E, suggesting that this antibody recognizes a discontinuous epitope not directly involved in gp120 binding. Moderate impairment of gp120 binding resulted from the insertion after amino acid residues 164 in the second immunoglobulin-like domain, where the epitopes for monoclonal antibodies MT151 and OKT4B were also mapped.     

Cellular factors influence the binding of HIV type 1 to cells

The goal of this study was to determine the importance of cellular factors for binding of HIV to cells. HIV primary isolates (PIs) produced in peripheral blood mononuclear cells (PBMCs) bound at relatively high levels to PBMCs but at low levels to cell lines, whereas T cell line-adapted (TCLA) virus produced in the H9 T cell line bound at high levels to both cell lines and PBMCs. Expression of CD4 in CD4-negative cells or blocking CD4 with antibody on CD4-positive cells did not affect virus binding. Blocking of gp120/gp41 with antibodies or a lack of expression of gp120/gp41 in virus particles also did not affect virus binding. However, the cell type from which virus was produced did affect virus binding. Thus, the binding pattern of TCLA virus shifted to that of a PI virus when produced in PBMCs. A PI binding pattern also occurred when a cloned TCLA virus (NL4-3) was produced in PBMCs, indicating that the virus-producing cell type has more of an effect on virus binding than the virus strain. These experiments show that both the virus-producing cell and the target cell have a major influence on HIV binding and suggest that host cell factors incorporated into virions are important for virus binding.     

Bispecific antibody targeting of human immunodeficiency virus type 1 (HIV-1) glycoprotein 41 to human macrophages through the Fc IgG receptor I mediates neutralizing effects in HIV-1 infection.

Human monocyte-derived macrophages that express the CD4 molecule and the Fc receptor for IgG (Fc gamma R) play a major role in the pathogenesis of human immunodeficiency virus (HIV) infection. To explore this possibility further, human monoclonal antibody to glycoprotein 41 (gp41) was produced, and a heterobifunctional antibody composed of F(ab') x F(ab')2 fragments of monoclonal anti-gp41 and anti-Fc gamma RI 22.2 were constructed. Both antibodies were analyzed for neutralizing effects, and the role of the CD4 molecule in HIV infection was studied with human monocyte-derived macrophages. The bispecific antibody exhibited strong neutralizing properties, in contrast to the monoclonal anti-gp41 antibody. Moreover, in the presence of monoclonal anti-Leu-3a antibody, viral production was completely inhibited. These findings demonstrate the necessity of the CD4 molecule in HIV infection of human macrophages and emphasize the usefulness of such heterobifunctional antibody directed to virus and monocyte-derived macrophage Fc receptors in prevention of HIV infection.     

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.     

Preparation and characterization of an intravenous solution of IgG from human immunodeficiency virus-seropositive donors

An intravenous solution of 99% pure globulin (hyperimmune IgG, HIVIG) was obtained from pooled plasma of selected human immunodeficiency virus (HIV-1)-seropositive asymptomatic donors with greater than 400 CD4+/microliters cells per microliter and a high titer of antibody to HIV-1 p24 protein. HIVIG had high titers of antibody to p24, glycoprotein 41 (gp41), and gp120, group-specific neutralizing activity, and binding to the gp120 hypervariable loop region. It inhibited syncytia formation. At low concentration, it enhanced viral production of HIV-1 in infected peripheral blood monocytes but was inhibitory at higher concentration. HIVIG directed group-specific antibody-dependent cellular cytotoxicity against HIV-infected targets. For a period of 6 to 28 months, plasma donors kept stable antibody titers and had a 1.0% decrease in CD4+ cells per month. One gram per kilogram HIVIG injected in two juvenile chimpanzees was well tolerated and did not transmit HIV, as measured by negative cell culture, IgM immune response to HIV proteins, and polymerase chain reaction. The mean half-life of HIV-1 p24 antibody was 15 days. These preliminary data suggest that HIVIG is a safe product suitable for clinical trial in HIV-1-infected individuals.     

Interactions of human antibodies,epitope exposure,antibody binding and neutralization of primary isolate HIV-1 virions

OBJECTIVE: Development of an effective HIV vaccine has been limited because of the inherent structural properties of the HIV envelope on native virions and the failure of the immune system to respond in an effective manner. Identification of the interactions of human antibodies with virions resulting in neutralization will facilitate vaccine design. DESIGN: Combinations of human monoclonal antibodies (hMAb) were studied for binding to and neutralization of primary isolate virions. METHODS: Virion binding and neutralization were measured using primary isolate virions. RESULTS: Antibodies and combinations of antibodies to epitopes exposed upon CD4 binding (CD4i) and V3 loop antibodies resulted in additive binding and neutralization of R5X4 virus. Antibodies did not bind to or neutralize R5 virus as well. The combination of V3 loop antibody with 2G12 resulted in enhanced neutralization and binding to the R5X4 isolate but not the R5 isolate. Preincubation of the R5X4 isolate with F240, a non-neutralizing anti-gp41 antibody, significantly enhanced binding and neutralization by CD4i hMAb and 2F5. F240 also enhanced the binding of 2F5 to the R5 isolate and the neutralization of the R5 isolate mediated by 2G12. CONCLUSIONS: Neutralizing epitopes are obscured on intact primary isolate virions and are dynamically exposed upon ligand (CD4) interactions. Interestingly, a non-neutralizing antibody to gp41 also increased binding and neutralizing activity of some hMAb that poorly neutralized R5 virus. These data suggest that non-neutralizing epitopes may be appropriate targets for vaccine design and epitope exposure should be considered in the development of immunotherapeutic strategies for HIV.     

Sequence similarities between human immunodeficiency virus gp41 and paramyxovirus fusion proteins

Cell fusion is a characteristic cytopathic effect induced by the human immunodeficiency virus (HIV) that leads to the formation of syncytia between infected lymphocytes. Although this process has been shown to occur following the specific binding of the 110-120 kD externalized envelope molecule of the virus with the CD4 glycoprotein, the region of the HIV envelope that directly mediates cell fusion is unknown. In an attempt to identify this fusion domain, we compared the amino acid sequences from the envelope molecules of several HIV isolates to the fusion proteins of paramyxoviruses. We found that the amino terminal region of the HIV transmembrane protein gp41 had a striking degree of similarity with the fusion domain of the respiratory syncytial virus. Moreover, similar sequences were noted in the fusion proteins of other paramyxoviruses and the transmembrane envelope proteins of a variety of lentiviruses suggesting that a functional relationship exists between these glycoproteins. This finding indicates that the amino terminal region of the HIV gp41 molecule may mediate cell fusion for this virus, and could be an important target in the design of immunologic strategies for the prevention of HIV infection in vivo.     

IgA antibodies of coeliac disease patients recognise a dominant T cell epitope of A-gliadin

BACKGROUND: In coeliac disease (CD) patients, the dominant DQ2-Alpha-I-gliadin peptide recognised by CD4 T cells is contained within peptide sequence 57-73 (p57-73) of Alpha-gliadin. This peptide sequence is also located within a 33-mer protease resistant gliadin fragment and therefore is likely to play an important role in the pathogenesis of CD. AIMS: Our aim was to determine whether a B cell epitope was present within the immunodominant T cell epitope of Alpha-gliadin and, if so, to elucidate its sequence and determine the importance of deamidation and/or modification of the amino acid at position 65 for IgA binding. PATIENTS AND METHODS: A cohort of CD patients, disease controls, and healthy individuals were examined. Serum IgA antibodies to the native and modified p57-73 fragment of Alpha-gliadin were analysed using enzyme linked immunosorbent assays. Peptide scanning experiments were further used to elucidate the B cell epitope. RESULTS AND CONCLUSION: IgA antibodies to p57-73 were found in 29/72 (40.2%) endomysial antibody positive patients, all of whom had CD. The peptide antibody appeared to be present when patients were on a diet containing gluten and declined on a gluten free diet. The p57-73 antibody was very specific for CD (98%) and had a sensitivity of 56%. The amino acid at position 65 was not important for IgA binding but was crucial for T cell recognition of p57-73. Pentapeptide PXPQP emerges as a potentially strong candidate for the IgA binding motif in this region of Alpha-gliadin. This study shows that a significant proportion of newly diagnosed CD patients have an antibody response to the immunodominant T cell epitope.     

Monoclonal anti-idiotypic antibody mimics the CD4 receptor and binds human immunodeficiency virus.

A monoclonal anti-idiotypic (anti-Id) antibody, HF1.7, was generated against anti-Leu-3a, a mouse monoclonal antibody (mAb) specific for the CD4 molecule on human helper/inducer T lymphocytes. The anti-Id nature of HF1.7 was demonstrated by the following properties. (i) It reacted in a solid-phase immunoassay with anti-Leu-3a and not with a panel of irrelevant mouse mAbs. (ii) It partially inhibited the binding of anti-Leu-3a to CD4+ T cells. (iii) It detected a common idiotype present on various anti-CD4 mAbs. Because the CD4 molecule represents the receptor site for human immunodeficiency virus (HIV), the etiologic viral agent of acquired immunodeficiency syndrome, we examined the ability of the anti-mAb HF1.7 to mimic CD4 and bind HIV. This anti-Id mAb reacted with HIV antigens in commercial HIV ELISAs and recognized HIV-infected human T cells but not uninfected cells when analyzed by flow cytofluorometry. Attesting further to the HIV specificity, the anti-Id mAb reacted with a recombinant gp160 peptide and a molecule of Mr 110,000-120,000 in immunoblot analysis of HIV-infected cell lysates. The anti-Id mAb also partially neutralized HIV infection of human T cells in vitro. These results strongly suggest that this anti-Id mAb mimics the CD4 antigenic determinants involved in binding to HIV.     

Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12

Human antibody 2G12 neutralizes a broad range of HIV-1 isolates. Hence, molecular characterization of its epitope, which corresponds to a conserved cluster of oligomannoses on the viral envelope glycoprotein gp120, is a high priority in HIV vaccine design. A prior crystal structure of 2G12 in complex with Man(9)GlcNAc(2) highlighted the central importance of the D1 arm in antibody binding. To characterize the specificity of 2G12 more precisely, we performed solution-phase ELISA, carbohydrate microarray analysis, and cocrystallized Fab 2G12 with four different oligomannose derivatives (Man(4), Man(5), Man(7), and Man(8)) that compete with gp120 for binding to 2G12. Our combined studies reveal that 2G12 is capable of binding both the D1 and D3 arms of the Man(9)GlcNAc(2) moiety, which would provide more flexibility to make the required multivalent interactions between the antibody and the gp120 oligomannose cluster than thought previously. These results have important consequences for the design of immunogens to elicit 2G12-like neutralizing antibodies as a component of an HIV vaccine.     

Enhanced HIV-1 neutralization by antibody heteroligation

Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.     

Combination of HIV-1-specific CD4 Th1 cell responses and IgG2 antibodies is the best predictor for persistence of long-term nonprogression

BACKGROUND: Strong T cell and antibody responses to human immunodeficiency virus (HIV), low virus production, and some genetic traits have been individually associated with nonprogression of HIV infection, but the best correlate with protection against disease progression remains unknown. METHODS: We prospectively followed 66 untreated long-term nonprogressors and analyzed relationships between HIV-1-specific CD4 T helper (Th) 1 and CD8 T cell responses and HIV-1-specific antibodies, HIV-1 RNA and proviral DNA loads, host genes, and CD4 Th1 cell counts at entry into the study and 4 years later. RESULTS: HIV-1 p24-specific CD4 Th1 cell proliferation, interferon (IFN)- gamma production, and IFN- gamma -producing cell frequencies at entry significantly and negatively correlated with HIV-1 RNA and proviral DNA loads and were independent of CD4 Th1 cell counts and host genes. HIV-1 Gag-specific IFN- gamma -producing CD8 T cell frequencies correlated with HIV-1 proviral DNA loads but not with RNA loads. Only high frequencies of HIV-1 p24-specific CD4 Th1 cells combined with HIV-1 gp41-specific IgG2 antibodies significantly predicted persistence of high CD4 Th1 cell counts. CONCLUSION: HIV-1-specific CD4 Th1 responses combined with IgG2 antibodies and IFN- gamma -producing CD4 Th1 cells are better predictors of long-term nonprogression than are virus parameters, host genes, or HIV-1-specific CD4 Th1 or CD8 T cell proliferation.     

The monoclonal CD4 antibody M-T413 inhibits cellular infection with human immunodeficiency virus after viral attachment to the cell membrane: an approach to postexposure prophylaxis.

Infectious cellular uptake of human immunodeficiency virus (HIV) is initiated by a complex sequence of interactions between the viral envelope gp120/gp41 complex and the cellular CD4 receptor resulting in the exposure of a hydrophobic region of gp41 that mediates the irreversible fusion of the virus with the cell membrane. Here we show that viral penetration into a susceptible cell can be inhibited by the high-affinity monoclonal CD4 antibody (CD4 mAb) M-T413 even when it is added as late as 30-120 min after the initial contact of virus with the cell membrane. Inhibition of infection was assessed by monitoring cultures for 34 days after exposure to virus using four different methods simultaneously, including detection of viral DNA by PCR. The interval during which HIV remains sensitive to postbinding neutralization by CD4 mAb depends on strain of virus and type of target cell. Preparations of recombinant soluble CD4 (and the immunoadhesin CD4-IgG1) were much less efficient when compared with mAb M-T413, particularly in blocking infection by fresh HIV-1 isolates. Also cellular transmission of HIV, as determined by syncytia formation within 24 hr, was prevented by mAb M-T413 when added within 45 min of contact of infected H9 cells with uninfected C8166 cells. Together with the favorable clinical experience obtained with CD4 mAbs as immunomodulatory drugs, these data suggest that infusion of CD4 mAb M-T413 may be a therapeutic modus for immediate prophylactic intervention after occupational exposure to HIV and for prevention of intrapartum mother-to-infant HIV transmission.     

Initial stages of HIV-1 envelope glycoprotein-mediated cell fusion monitored by a new assay based on redistribution of fluorescent dyes.

Membrane fusion is an essential step in the infection of permissive cells with human immunodeficiency virus (HIV). Infected cells frequently fuse with each other, and then progress to form multinucleated giant cells (syncytia). To gain insight into mechanisms of HIV env-mediated membrane fusion, we developed a new assay for studying the initial events. The assay is based on the redistribution of fluorescent markers between membranes and cytoplasm of adjacent cells examined by means of fluorescence video microscopy. Membrane fusion between HIV-1 envelope glycoprotein (gp120/41) expressing effector cells and CD4+ target cells was observed 90 min after the association of cells, whereas the first syncytia only became apparent after 5 h. Moreover, membrane fusion events were observed under conditions where no syncytia were detected, for example, when the effector:target cell ratio was greater than 100:1, or less than 1:100. A significant number of cells with fused membranes were not involved in the syncytia. In order to determine whether quantitative differences in receptor expression might influence the extent of membrane fusion, we used laboratory-selected variants of CEM cells that differ in their expression of CD4. We found that CD4 is required on the target membrane for HIV env-mediated membrane fusion, but its extent is only partially dependent on CD4 surface concentration. The ability of those CEM variants to take part in HIV env-mediated membrane fusion did not correlate with their capacity to form syncytia. These findings indicate that additional steps are needed to form syncytia after membrane fusion.     

Designing immunogens to elicit broadly neutralizing antibodies to the HIV-1 envelope glycoprotein

To date HIV-1 vaccines have not been able to elicit potent, long lasting, and broadly neutralizing antibodies to the virus. Our knowledge of HIV envelope glycoprotein (Env) structure/function and the existence of a handful of broadly neutralizing antibodies is guiding rational immunogen design. We review here the potential targets on the HIV Env (the glycan shield, the CD4 binding site, the coreceptor binding site, Env fusion intermediates, and the membrane proximal region) and their associated rational immunogen design strategies. Moreover, we discuss immune dampening and immune refocusing strategies designed to counter immunodominant, decoy responses generated by the virus. In this regard, an immunogen design strategy of "in vitro de-evolution" is presented, which begins to distill the HIV Env to its most critical, core functional domains. While we are beginning to have some understanding as to where we would like out immune system to go, we find that our immune repertoire may actually have limits that preclude successful completion of the task at hand. The repertoire limits appear to be a byproduct of autoantibody tolerance mechanisms and the complex structural requirements for effective, potent broadly neutralizing antibodies. Nevertheless, the hope is that through novel insights and creative solutions that we will be able to design immunogens capable of eliciting broadly neutralizing antibodies to the HIV envelope glycoprotein.     

CD4 peptide-protein conjugates, but not recombinant human CD4, bind to recombinant gp120 from the human immunodeficiency virus in the presence of serum from AIDS patients.

Sera from human immunodeficiency virus-positive (HIV+; Walter Reed stage 6) individuals inhibit the interaction between recombinant human CD4 and recombinant gp120 from HIV (rCD4 and rgp120, respectively), thereby interfering with the ability of soluble rCD4 to block infection with HIV or rCD4-toxin conjugates to kill HIV-infected cells. In this report we demonstrate that the inhibitory activity of such sera is caused primarily by anti-gp120 antibodies that do not recognize the CD4 interaction site on gp120. To circumvent the problem of inhibition, we have generated a construct containing a peptide of CD4 (residues 41-84) conjugated to ovalbumin (three to five peptides per molecule). This multivalent conjugate binds to rgp120 and binding is not inhibited by antibodies in HIV+ sera.     

Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.

Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.     

Cross-Reactive Human IgM-Derived Monoclonal Antibodies that Bind to HIV-1 Envelope Glycoproteins

Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs) isolated from humans with HIV-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs) are immunoglobulin (Ig) Gs (IgGs) and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs) are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs) of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further characterize conserved non-neutralizing, weakly neutralizing or enhancing epitopes and modify or remove them from candidate vaccine immunogens.