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Hermann Katinger

Publications and source records attributed to Hermann Katinger.

At least 73 records · Page 4Linked to original sources
PubMed2003

Distinct host range of influenza H3N2 virus isolates in Vero and MDCK cells is determined by cell specific glycosylation pattern.

Influenza A viruses were isolated in Vero, MDCK cells and chicken embryos. In contrast to MDCK-derived variants all H3N2 isolates obtained in Vero cells neither agglutinated chicken erythrocytes nor grew in chicken eggs. These host range differences of H3N2 Vero and MDCK isolates were noticed even in the absence of amino acid substitutions in the HA1 molecule. Evaluation of HA glycosylation pattern by treatment with endoglycosidases H and F revealed that Vero-variants contained more oligosaccharides of the high mannose type than did the corresponding MDCK-isolates. Removal of some mannose residues from the non-reducing termini of the carbohydrates by exomannosidase treatment resulted in the ability of Vero-isolates to agglutinate chicken erythrocytes. Glycosylation pattern and properties of H3N2 viruses grown in Vero cells were close to those of viruses grown in human kidney epithelial cells, whereas the H1N1 variants isolated from Vero, MDCK cells or eggs did not differ in agglutination properties, carbohydrate composition or ability to infect eggs.

Animals↗
PubMed2003

Post-exposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques.

BACKGROUND: The majority of infants infected through maternal transmission acquire the virus during birth or postpartum through breastfeeding: mucosal exposure is considered to be a major route of infection. OBJECTIVES: To develop passive immunization with human neutralizing monoclonal antibodies (mAbs) against mother-to-child transmission of HIV during delivery and through breastfeeding. DESIGN: An oral challenge model in newborn rhesus macaques mimicked peri- and postpartum virus transmission. METHODS: Neonatal rhesus macaques were challenged orally with the highly pathogenic, chimeric simian-human immunodeficiency virus SHIV89.6P and given post-exposure prophylaxis with a quadruple combination of neutralizing human mAbs, IgG1b12, 2G12, 2F5, and 4E10, directed against conserved epitopes of HIV envelope glycoproteins. Control animals were virus challenged but left untreated. All infants were followed prospectively for signs of viremia and immunodeficiency. RESULTS: Two out of four macaque infants treated with neutralizing mAbs showed no evidence of infection; the other two maintained normal CD4 T cell counts. In contrast, all control animals became highly viremic and had profound CD4 T cell losses; three out of four died from AIDS within 1.5-6 weeks of the challenge. CONCLUSIONS: Passive immunization with this quadruple neutralizing mAbs combination may represent a promising approach to prevent peri- and postnatal HIV transmission. Furthermore, the epitopes recognized by the four neutralizing mAbs are key determinants to achieve complete protection and represent important targets against which to develop active, antibody-response-based AIDS vaccines.

Animals↗
PubMed2003

Primary African HIV clade A and D isolates: effective cross-clade neutralization with a quadruple combination of human monoclonal antibodies raised against clade B.

We investigated the ability of several human neutralizing monoclonal antibodies (nmAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade A and D isolates as single agents and in combinations. All four primary HIV clade A isolates and five primary HIV clade D isolates tested were neutralized >99% by the quadruple combination of nmAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognize conserved epitopes on HIV-1 envelope (Env), resulting in strong cross-clade neutralization. Previously, we showed synergistic neutralization of primary HIV-1 clade C isolates in vitro by the same nMAb combination. We and others also showed neutralization of primary HIV clade B strains. Together, our data show that the quadruple combination of mAbs effectively neutralized primary HIV clade A, B, C, and D isolates.

Africa↗
PubMed2003

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.

Animals↗
PubMed2003

Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12.

We have previously generated human monoclonal anti-human immunodeficiency virus type 1 (anti-HIV-1) antibodies 2F5IgG and 2G12IgG with an exceptional cross-clade neutralizing potential. 2F5IgG and 2G12IgG passively administrated to macaques were able to confer complete protection from both intravenous and mucosal challenge with pathogenic HIV-simian immunodeficiency virus chimeric strains and have shown beneficial effects in a phase-1 clinical trial. We now class-switched 2F5 and 2G12 to the immunoglobulin M (IgM) or IgA isotype, to enforce features like avidity, complement activation, or the potential to neutralize mucosal transmission. For this purpose we expressed functional polymeric 2F5 and 2G12 antibodies in CHO cells and evaluated their anti-HIV-1 activity in vitro. The class switch had a strong impact on the protective potential of 2F5 and 2G12. 2G12IgM inhibited HIV-1 infection of peripheral blood mononuclear cell cultures up to 28-fold-more efficiently than the corresponding IgG and neutralized all of the primary isolates tested. The 2F5 and 2G12 antibodies of all isotypes were able to interact with active human serum to inhibit viral infection. Furthermore, we demonstrated that polymeric 2F5 and 2G12 antibodies but not the corresponding IgGs could interfere with HIV-1 entry across a mucosal epithelial layer in vitro. Although polymeric 2F5 antibodies had only limited potential in the standard neutralization assay, the results from the mucosal assay suggest that 2F5 and 2G12 antibodies may have a high potential to prevent natural HIV-1 transmission in vivo.

Animals↗
PubMed2002

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

Antibody Affinity↗
PubMed2002

Antiviral activity of the neutralizing antibodies 2F5 and 2G12 in asymptomatic HIV-1-infected humans: a phase I evaluation.

BACKGROUND: The human monoclonal antibodies (MAbs) 2F5 and 2G12 were identified to be two of the most potent neutralizing antibodies against HIV-1. In a first human study they have been shown to be safe after repeated intravenous infusions to asymptomatic HIV-1-infected individuals. However, the antiviral effects of antibody treatment have not been fully analyzed in this first clinical trial. METHODS: The aim of the present study was to gain a preliminary insight into the antiviral effects of 2F5 and 2G12 in humans. For this purpose, plasma samples obtained from the previous phase I study were studied for RNA copy numbers by reverse transcriptase-polymerase chain reaction. As a measure for activation of complement levels of the major complement factor C3 were measured by enzyme-linked immunosorbent assay. Flow cytometry was used to study T-lymphocyte counts and the amount of infected peripheral blood mononuclear cells (PBMC) was determined by co-culture with uninfected donor PBMC. Virus escape from antibody neutralization was determined in vitro in a PBMC neutralization assay. RESULTS: Transient reduction in viral loads was observed in five of seven patients. Vigorous complement activation was observed directly after HIV-specific antibody infusions. The number of infective peripheral blood mononuclear cells was reduced in some patients whereas CD4+ T-lymphocyte counts and CD4+/CD8+ ratios were transiently increased in all patients. Virus escape occurred only against 2G12. CONCLUSIONS: Analysis of disease progression markers indicate that antibody therapy may have antiviral effects. These findings suggest that neutralizing antibodies should be further evaluated as an alternative therapeutic approach in HIV-1 disease.

Adult↗
PubMed2002

Optimization of recombinant protein expression level in Escherichia coli by flow cytometry and cell sorting.

Overexpression of recombinant proteins in Escherichia coli often leads to a severe growth retardation of the host cells. The phage T7 promoter phi10 in a pET vector was utilized to express human superoxide dismutase. Induction with IPTG lead to an increase in protein content and cell size and a termination of cell division, due to the deviation of the general metabolic fluxes from all cellular processes to plasmid maintenance and foreign protein synthesis. To generate promoter mutants which are better tolerated by the host cells we constructed a random mutation library by PCR with degenerated primers in a part of the promoter involved in the binding to the RNA polymerase and the initiation of transcription. This library was sorted by flow cytometry for cells with a lower total protein content as an indicator for continued cell replication and hence a less severe stress situation. The clones obtained had a similar SOD production compared to the original strain, but were able to reach higher densities in a batch culture, which resulted in a higher total yield.

Bacterial Proteins↗
PubMed2002

Anti-idiotypic antibody Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal antibody 2F5.

Anti-idiotypic antibodies directed against potentially neutralizing anti-HIV-1 antibodies may mimic epitopes on gp41 otherwise cryptic to the immune system. This study reports the generation of murine monoclonal antibody Ab2/3H6 blocking the binding of human Ab1 2F5 to the synthetic epitope and to gp160 in an enzyme-linked immunosorbent competition assay. Ab2/2H6 diminished the neutralizing potency of 2F5 in an in-vitro neutralization assay. Ab2/3H6 Fab fragments were capable of inducing neutralizing immune and 2F5-specific responses in B6D2F1 mice applying a simple prime-boost regimen of immunization.

Animals↗
PubMed2002

Age-related alterations in the protein expression profile of C57BL/6J mouse pituitaries.

The aim of our study was to monitor the protein expression profile in pituitary glands of healthy C57BL/6J mice during aging. Pituitary glands of 4-week old (immature), 3-month old (mature), and >25-month old mice were analysed by proteomic tools such as two-dimensional electrophoresis and N-terminal micro-sequencing. A change was detected in the expression of growth hormone after sexual maturation. Our particular interest, however, was directed against up-regulated proteins in the old pituitary glands, which are proposed to be involved in the process of neuroendocrine aging. Among these proteins, the expression of glutathione-S-transferase (GST) and apolipoprotein A-1 were increased in old pituitaries. Furthermore, ubiquitin carboxyl-terminal hydrolase (UCH-L1) was significantly up-regulated in senescent C57BL/6J mouse pituitaries. Since only the rat homologue was known, we isolated and analysed the mouse UCH-L1 sequence. Since GST is involved in antioxidative defence and UCH-L1 is part of the ubiquitin/proteasome system, which is responsible for the removal of damaged proteins, these results suggest increased oxidative burden and an increased activity of the ubiquitin system.

Aging↗
PubMed2002

Long term stability of rh-Cu/Zn-superoxide dismutase (SOD)-liposomes prepared by the cross-flow injection technique following International Conference on Harmonisation (ICH)-guidelines.

The current market position of liposomes as drug carriers is still being discussed with regard to large scale production, product characterisation and the stability of the dispersions. In this study, long term stability of liposomal suspensions with encapsulated rh-Cu/Zn-superoxide dismutase was tested according to the International Conference on Harmonisation (ICH) recommendations. The guidelines of the ICH provide general requirements for stability testing for registration and export in particular. The Institute of Applied Microbiology has examined a process to produce large amounts of pharmaceutical-grade liposomes for the treatment of inflammatory diseases by topical application. For the evaluation of its long-term storage stability, liposomal stability and protein stability were tested under appropriate conditions. Therefore, size alterations of the vesicles, protein release and protein activity were evaluated. During the observation period, neither significant alterations of the liposomes nor any protein degradation could be detected. In the light of these findings our liposomal formulations seem to provide chemical, physical and biological stability according to the definitions of the ICH. Appropriate lipid compounds and environmental factors, in combination with an optimised process and adequate storage conditions, facilitate the production of liposome dispersions suitable as drug carriers on the market.

Drug Stability↗
PubMed2002

Liposomes produced in a pilot scale: production, purification and efficiency aspects.

Intensive efforts have been made to establish a novel, scalable liposomal preparation technique suitable for the entrapment of even large proteins into liposomes. We have developed a new technique based on the principles of the ethanol injection technique. Herein, the principal item is the crossflow injection module, specifically designed for this purpose. This unit has the benefit of defined and characterized injection streams and permits liposome manufacture regardless of production scale, as scale is determined only by the free disposable vessel volumes. Previous publications demonstrated that the crossflow injection technique that we have developed meets all of the above-mentioned requirements. The present paper describes the entire three-step production process, consisting of encapsulation, separation of non-entrapped protein by continuous crossflow filtration, and retrieval of rh-Cu/Zn-SOD by additional filtration. Results of consecutive lots were compared, based on well-defined quality criteria.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2002

The crossflow injection technique: an improvement of the ethanol injection method.

A novel scalable liposome preparation technique for pharmaceutical application is presented. Previous experiments have shown that the concept of continuous crossflow injection is a promising approach. For the characterization of the process, we focus on the influencing parameters like the lipid concentration, the injection hole diameter, the injection pressure, the buffer flow rate, and system performance. These experiments demonstrate that the injection hole diameter and the system performance do not influence the vesicle forming process and that a minimum of buffer flow rate is required to affect batch homogeneity. In contrast, strongly influencing parameters are lipid concentration in combination with increasing injection pressures. After exceeding the upper pressure limit of the linear range, where injection velocities remain constant, the vesicle batches are narrowly distributed, also when injecting higher lipid concentrations. Reproducibility and scalability data show similar results with respect to vesicle size and size distribution and demonstrate the stability and robustness of the novel continuous liposome preparation technique.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

Enhanced protein loading into liposomes by the multiple crossflow injection technique.

Methods for encapsulation of a drug into liposomes should preferably result in a high encapsulation efficiency and a high encapsulation capacity. Our studies were focussed on the establishment of an efficient encapsulation procedure of the radical scavenging protein, rh-Cu/Zn-SOD, into liposomes with the cross flow injection method. Limitations to increase the encapsulation efficiency are caused by the enclosed aqueous volume, by the lipid concentration, the aspired vesicle size and the final ethanol concentration. Our research was performed to maximize the encapsulation following several strategies of injecting higher lipid concentrations into the aqueous phase. The one way triple technique, a sophisticated preparation procedure is presented, which enables three times higher encapsulation rates in comparison to standard procedures. Additionally, scalability studies demonstrate reproducibility independent of the preparation volume. Vesicle size distribution and encapsulation efficiency remain constant. Furthermore, special attention is paid on reproducibility of prepared liposomes, scale-up and on long term stability of the lipid vesicles.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120.

We have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.

Antibodies, Monoclonal↗
PubMed2002

The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha1-->2 mannose residues on the outer face of gp120.

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N-->A substitution produced significant decreases in 2G12 binding affinity to gp120(JR-CSF). Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120(JR-CSF). Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Man(alpha)1-->2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Man(alpha)1-->2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Man(alpha)1-->2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.

Amino Acid Sequence↗