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Biomedical subjects

P W Parren

Publications and source records attributed to P W Parren.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.

We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity.

AIDS Vaccines↗
PubMed2001

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Adult↗
PubMed2001

Crystallization and preliminary structure determination of an intact human immunoglobulin, b12: an antibody that broadly neutralizes primary isolates of HIV-1.

An intact human immunoglobulin with a full-length hinge has been crystallized for the first time in a form in which all of the Ig domains are ordered. The IgG1 antibody b12 is one of only three known monoclonal antibodies described that potently neutralize a broad range of HIV-1 primary isolates. It binds to an epitope overlapping the conserved CD4 binding site on the viral surface antigen gp120. Hexagonal crystals corresponding to space group R32 were grown from 0.8 M ammonium sulfate, with unit-cell parameters a = b = 271.3, c = 175.2 A and one molecule per asymmetric unit. The crystals diffract to 2.8 A and a preliminary molecular-replacement solution indicates that all 12 Ig domains of the antibody can be resolved.

Crystallization↗
PubMed2001

Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12.

Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.

Antibodies, Monoclonal↗
PubMed2001

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro.

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.

Administration, Intravaginal↗
PubMed2001

Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41.

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.

Amino Acid Motifs↗
PubMed2001

Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1.

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in FcgammaR binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either FcgammaR or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.

Antibody-Dependent Cell Cytotoxicity↗
PubMed2001

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.

AIDS Vaccines↗
PubMed2001

gp120: Biologic aspects of structural features.

HIV-1 particles are decorated with a network of densely arranged envelope spikes on their surface. Each spike is formed of a trimer of heterodimers of the gp120 surface and the gp41 transmembrane glycoproteins. These molecules mediate HIV-1 entry into target cells, initiating the HIV-1 replication cycle. They are a target for entry-blocking drugs and for neutralizing Abs that could contribute to vaccine protection. The crystal structure of the core of gp120 has been recently solved. It reveals the structure of the conserved HIV-1 receptor binding sites and some of the mechanisms evolved by HIV-1 to escape Ab responses. The gp120 consists of three faces. One is largely inaccessible on the native trimer, and two faces are exposed but apparently have low immunogenicity, particularly on primary viruses. We have modeled HIV-1 neutralization by a CD4 binding site monoclonal Ab, and we propose that neutralization takes place by inhibition of the interaction between gp120 and the target cell membrane receptors as a result of steric hindrance. Knowledge of gp120 structure and function should assist in the design of new drugs as well as of an effective vaccine. In the latter case, circumventing the low immunogenicity of the HIV-1 envelope spike is a major challenge.

AIDS Vaccines↗
PubMed2000

A structure-based approach to a synthetic vaccine for HIV-1.

The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --> O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with >1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.

AIDS Vaccines↗
PubMed2000

Absence of specific mucosal antibody responses in HIV-exposed uninfected sex workers from the Gambia.

OBJECTIVES: Specific antibodies to HIV envelope that inactivate virus at the mucosal surfaces involved in sexual contact are of interest for the design of a vaccine against HIV-1. It has been suggested that, in frequently HIV-exposed but uninfected individuals, HIV-specific mucosal antibody responses may exist and play a role in resistance against HIV. This study investigated HIV-1 envelope specific mucosal antibody responses in HIV-resistant sex workers in west Africa. METHODS: A group of 26 exposed uninfected female commercial sex workers from the Gambia, who have had repeated exposures to HIV-1 and HIV-2 were studied. We assessed the presence of vaginal IgA and IgG in vaginal swabs against a range of HIV-1 and HIV-2 envelope presentations and performed HIV-1 neutralization assays. RESULTS: No significant vaginal IgA or IgG responses against HIV-1 or HIV-2 were detected, and none of the vaginal secretions tested displayed any HIV-1 neutralizing activity. CONCLUSION: Vaginal antibody responses against HIV were not found in Gambian sex workers who resist HIV infection. Resistance against HIV infection can therefore occur in the absence of specific antibodies against HIV at the genital mucosa. A protective role for HIV-envelope specific IgA in resistance against HIV-1 infection in exposed uninfected individuals as reported in the literature is uncertain.

Antibody Formation↗
PubMed2000

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A.

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.

Amino Acid Motifs↗