Biomedical subjects

Christoph Grundner

Publications and source records attributed to Christoph Grundner.

9 recordsLinked to original sources

PubMed2005

Analysis of the neutralizing antibody response elicited in rabbits by repeated inoculation with trimeric HIV-1 envelope glycoproteins.

The elicitation of broadly neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, gp120 and gp41, remains a major challenge. Attempts to utilize monomeric gp120 as an immunogen to elicit high titers of neutralizing antibodies have been disappointing. Envelope glycoprotein constructs that better reflect the trimeric structure of the functional envelope spike have exhibited improved immunogenicity compared with monomeric gp120. We have described soluble gp140 ectodomain constructs with a heterologous trimerization motif; these have previously been shown to elicit antibodies in mice that were able to neutralize a number of HIV-1 isolates, among them primary isolate viruses. Recently, solid-phase proteoliposomes retaining the envelope glycoproteins as trimeric spikes in a physiologic membrane setting have been described. Here, we compare the immunogenic properties of these two trimeric envelope glycoprotein formulations and monomeric gp120 in rabbits. Both trimeric envelope glycoprotein preparations generated neutralizing antibodies more effectively than gp120. In contrast to monomeric gp120, the trimeric envelope glycoproteins elicited neutralizing antibodies with some breadth of neutralization. Furthermore, repeated boosting with the soluble trimeric formulations resulted in an increase in potency that allowed neutralization of a subset of neutralization-resistant HIV-1 primary isolates. We demonstrate that the neutralization is concentration-dependent, is mediated by serum IgG and that the major portion of the neutralizing activity is not directed against the gp120 V3 loop. Thus, mimics of the trimeric envelope glycoprotein spike described here elicit HIV-1-neutralizing antibodies that could contribute to a protective immune response and provide platforms for further modifications to improve the efficiency of this process.

Amino Acid Sequence↗

PubMed2005

Mycobacterium tuberculosis protein tyrosine phosphatase PtpB structure reveals a diverged fold and a buried active site.

Intracellular pathogenic bacteria manipulate host signal transduction pathways to facilitate infection. Mycobacterium tuberculosis protein tyrosine phosphatases (PTPs) PtpA and PtpB are thought to be secreted into host cells and interfere with unidentified signals. To illuminate the mechanisms of regulation and substrate recognition, we determined the 1.7 A resolution crystal structure of PtpB in complex with the product phosphate. The protein adopts a simplified PTP fold, which combines features of the conventional PTPs and dual-specificity phosphatases. PtpB shows two unusual elaborations--a disordered, acidic loop and a flexible, two-helix lid that covers the active site--that are specific to mycobacterial orthologs. Biochemical studies suggest that substrate mimicry in the lid may protect the phosphatase from oxidative inactivation. The insertion and deletion of large structural elements in PtpB suggest that, outside the active site module, the PTP family is under unusual selective pressure that promotes changes in overall structure.

Binding Sites↗

PubMed2005

Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains.

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.

ATP-Binding Cassette Transporters↗

PubMed2005

Structure/function studies of Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis.

Many bacterial species express 'eukaryotic-like' Ser/Thr or Tyr protein kinases and phosphatases that are candidate mediators of developmental changes and host/pathogen interactions. The biological functions of these systems are largely unknown. Recent genetic, biochemical and structural studies have begun to establish a framework for understanding the systems for Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis (Mtb). Ser/Thr protein kinases (STPKs) appear to regulate diverse processes including cell division and molecular transport. Proposed protein substrates of the STPKs include putative regulatory proteins, as well as six proteins containing Forkhead-associated domains. Structures of domains of receptor STPKs and all three Mtb Ser/Thr or Tyr phosphatases afford an initial description of the principal modules that mediate bacterial STPK signaling. These studies revealed that universal mechanisms of regulation and substrate recognition govern the functions of prokaryotic and eukaryotic STPKs. Several structures also support novel mechanisms of regulation, including dimerization of STPKs, metal-ion binding to PstP and substrate mimicry in PtpB.

Bacterial Proteins↗

PubMed2004

Factors limiting the immunogenicity of HIV-1 gp120 envelope glycoproteins.

Efficient immune responses to HIV-1 gene products are essential elements to the development and design of an effective vaccine. Ideally, both humoral and cellular responses will be optimally elicited. It is therefore important to elucidate any factors that might limit the immunogenicity of HIV-1 proteins that are likely to be included in an effective vaccine. Since the HIV-1 exterior envelope glycoprotein gp120 is a major target for neutralizing antibodies, it is a virtual certainty that this gene product will be a component of any vaccine that seeks to elicit neutralizing antibody responses from the host humoral immune system. We report here the testing of several HIV-1 gp120 variants derived from a primary isolate that appears deficient in eliciting immune responses at both the level of CD4+ help and consequently in the generation of high-affinity IgG antibody responses in small animals. Factors limiting an effective immune response include (a) envelope glycoprotein strain variation decreasing functional T-cell help, (b) alteration of the glycosylation patterns of gp120 by expression in different cell types, and (c) the native structure of gp120 itself, which may limit the elicitation of effective T-cell help during natural infection or during parenteral immunization in adjuvant. Such limiting factors and others should be considered in the design and testing of gp120-based immunogens in small animals and possibly in primates as well.

AIDS Vaccines↗

PubMed2003

Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition.

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.

Binding Sites↗

PubMed2003

Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120.

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.

Amino Acid Sequence↗

PubMed2003

Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1.

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.

Antibodies, Monoclonal↗

PubMed2002

Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins.

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160DeltaCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160DeltaCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160DeltaCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.

Antigens, Viral↗