Human Immunoglobulin Preparations Research Articles

Determination of slime-producing S. epidermidis specific antibodies in human immunoglobulin preparations and blood sera by an enzyme immunoassay : Correlation of antibody titers with opsonic activity and application to preterm neonates

Slime-producing Staphylococcus epidermidis is responsible for severe infections in immunocompromised patients and, particularly, in premature infants who are transiently deficient in IgG. A sulfated polysaccharide with molecular mass of 20-kDa (20-kDa PS) has been recognized as the major polysaccharide component and antigenic determinant of S. epidermidis extracellular slime layer. The presence of adequate amounts of antibodies to 20-kDa PS in patients’ sera would be of importance to prevent or treat slime-producing S. epidermidis bacteremia. Administration of intravenous immunoglobulin (IVIG) is considered to be a reasonable IgG replacement therapy and has been widely used to prevent or treat neonatal sepsis. Clinical trials have shown conflicting results on the efficacy of IVIGs and this phenomenon has been attributed to the variability of IVIG preparations in the content and opsonic activity of IgG against microorganisms of clinical importance. Monitoring of antibodies to distinct bacterial macromolecules, which are species-specific and responsible for bacterial infections, has not been performed previously. A highly precise and repeatable enzyme immunoassay was developed to determine quantitatively the levels of antibodies against the 20-kDa PS of S. epidermidis slime. The amount of 20-kDa PS specific antibodies found in 27 lots of an IVIG preparation (Sandoglobulin®) correlated well with their in vitro opsonic activity against slime-producing S. epidermidis. The majority of lots (75%) having titers higher than 200 units/ml showed significant opsonic activity (50–75%) towards slime-producing S. epidermidis. Sandoglobulin® lots with titers higher than 200 units/ml of 20-kDa PS specific IgG were administered as a prophylactic agent to low-birth weight (lower than 1700 g) preterm neonates immediately after birth. The levels of total and 20-kDa PS specific IgG in neonates’ blood sera were significantly higher than those found in the control group, even 10 days after the last infusion. The rate of slime-producing S. epidermidis bacteremia in neonates who received IVIG was also considerably lower than those in the control group. The results of this study suggest that specific IgG titers estimated by the developed enzyme immunoassay may well be indicative of the IVIG opsonic activity against slime-producing S. epidermidis. Furthermore, administration of Sandoglobulin® with titers higher than a cut-off value of 200 units/ml may significantly protect preterm neonates against slime-producing S. epidermidis bacteremia.

Neutralizing antibodies to granulocyte–macrophage colony‐stimulating factor, interleukin‐1α and interferon‐α but not other cytokines in human immunoglobulin preparations

Human immunoglobulin preparations are used therapeutically for various disorders. Such therapy is generally safe but adverse effects occasionally occur in recipients. It has been suggested that antibodies to cytokines present in clinical immunoglobulin products may contribute to undesirable effects in recipients. Therefore, we investigated intravenous and intramuscular immunoglobulin products for the presence of cytokine-specific neutralizing antibodies. Using validated bioassays, we detected neutralizing activity against human granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha2a (IFN-alpha2a) and interleukin-1alpha (IL-1alpha) in immunoglobulin products. We found no neutralization of granulocyte colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12, tumour necrosis factor-alpha, oncostatin M (OSM) and IFN-gamma. Most batches which neutralized IFN-alpha2a activity also neutralized other IFN-alpha subtypes, IFN-omega and IFN-beta. Most products (94%) neutralized the biological activity of GM-CSF. No correlation between batches and their ability to neutralize bioactivities of GM-CSF, IFN-alpha2a and IL-1alpha was found. This neutralizing activity could be traced to plasma pools used for manufacture of immunoglobulins. The neutralization was mediated by specific cytokine antibodies contained within immunoglobulin products as it was present in specific immunoglobulin G (IgG) fractions eluted from cytokine affinity chromatography columns. Specific binding of such IgG fractions to cytokines in immunoblots and in enzyme-linked immunosorbent assays (ELISAs) was observed. This contrasts with the broad non-specific recognition of cytokine proteins observed using unfractionated immunoglobulins in ELISAs. This is the first comprehensive study showing the presence of neutralizing antibodies against GM-CSF, IL-1alpha, or IFN-alpha2a in immunoglobulin products.

Human Intravenous Immunoglobulin Preparation and Virus Inactivation by Pasteurization and Solvent Detergent Treatment

ABSTRACT Human intravenous immunoglobulin (IVIG) solutions were prepared by two different methods and compared to each other. The crude immunoglobulin fraction obtained from Cohn-Oncley fractionation of plasma was further purified and subjected to virus inactivation, either by polyethylene glycol precipitation and pasteurization at 60°C for 10 hours, or by ion exchange chromatography and solvent/detergent treatment.

An enzyme immunoassay to determine the levels of specific antibodies toward bacterial surface antigens in human immunoglobulin preparations and blood serum

Human polyvalent intravenous immunoglobulin (IVIG) preparations are used as a complementary aid to the proper antimicrobial treatment of severely septic patients in intensive care units (ICUs) and/or as a prophylactic agent to immunocompromized hosts, particularly prone to bacterial infections. There is skepticism about the usefulness of IVIGs since it is not known whether their administration ensures the enhancement of humoral immune responses by providing a sufficient amount of specific antibodies towards the specified bacterial pathogen to be treated. In this report, a simple and reproducible enzyme-linked immunosorbent assay for determining the content of specific antibodies against bacterial surface antigens in commercially available IVIG preparations is described. The method is also easily applied to determine the amount of bacterial antibodies in blood serum. The levels of specific antibodies toward Gram positive and negative pathogenic isolates often encountered in ICUs were estimated in two IVIG (Sandoglobulin® and Gamimmune®) preparations. Significant differences regarding the content of antibodies to certain clinically bacterial isolates were identified not only between the two IVIG preparations tested, but also among various lots from each IVIG preparation. No significant variation ( P≤0.001) among the bottles derived from the same lot was determined in both preparations. The variation in the levels of specific antibodies in IVIG preparations may be attributed to differences between the donor pools as well as the manufacturing procedure. Application of the method to patients with primary immune deficiencies showed that infusion of highly reactive IVIG preparations enhanced significantly their humoral response toward various pathogens. The results of this study suggest that the content determination of pathogen-specific antibodies in IVIG preparations before administration may be of great importance for treating bacterial infections.

Synergical activities of sulfonated human immunoglobulin preparations and the various antibiotics against penicillin resistant Streptococcus pneumoniae

Synergical activities of sulfonated human immunoglobulin preparations and the various antibiotics against penicillin resistant <I>Streptococcus pneumoniae</I>

Synergistic Neutralization of Simian-Human Immunodeficiency Virus SHIV-vpu+by Triple and Quadruple Combinations of Human Monoclonal Antibodies and High-Titer Anti-Human Immunodeficiency Virus Type 1 Immunoglobulins

We have tested triple and quadruple combinations of human monoclonal antibodies (MAbs), which are directed against various epitopes on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, and a high-titer anti-HIV-1 human immunoglobulin (HIVIG) preparation for their abilities to neutralize a chimeric simian-human immunodeficiency virus (SHIV-vpu+). This virus encodes the HIV-1 strain IIIB env, tat, rev, and vpu genes. The quantitative nature of the Chou-Talalay method (Adv. Enzyme Regul. 22:27-55, 1984) allows ranking of various combinations under identical experimental conditions. Of all triple combinations tested, the most potent neutralization was seen with MAbs 694/98D plus 2F5 plus 2G12 (directed against domains on V3, gp41, and gp120, respectively) as measured by the total MAb concentration required to reach 90% neutralization (90% effective concentration [EC90], 2.0 microg/ml). All triple combinations involving MAbs and/or HIVIG that were tested yielded synergy with combination index values of < 1; the dose reduction indices (DRIs) ranged from 3.1 to 26.2 at 90% neutralization. When four MAbs (the previous three plus MAb F105, directed against the CD4 binding site) were combined, higher neutralization potency (EC90 1.8 microg/ml) and a higher degree of synergy compared to any triple combination were seen. The mean DRIs of the quadruple combination were approximately twice that of the most synergistic triple combination. We conclude that human MAbs targeting different HIV-1 envelope glycoprotein epitopes exhibit strong synergy when used in combination, a fact that could be exploited clinically for passive immunoprophylaxis against HIV-1.

Antibody to Granulocyte-Macrophage Colony-Stimulating Factor Is a Dominant Anti-Cytokine Activity in Human IgG Preparations

AbstractPharmaceutical preparations of normal human immunoglobulin (IgG) are known to contain high-avidity and neutralizing antibodies (Ab) to the cytokines interleukin (IL)-1α, IL-6, and interferon (IFN)α. To test for other cytokine Ab, 23 batches of IgG were tested for saturable binding to eight 125I-labeled recombinant cytokines. All batches bound granulocyte-macrophage colony-stimulating factor (GM-CSF) with high avidity (Kav ≈ 10 pmol/L) and capacities of up to 5 μmol GM-CSF/mol IgG. Only 1 of 15 batches bound IL-5, also with high avidity, whereas 13 of 15 batches bound to IL-10 but with lower capacities and avidities. None of the IgG preparations bound IL-1 receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, or G-CSF. Cross-binding and absorption analyses revealed identical or slightly stronger binding of recombinant GM-CSF, IL-5, and IL-10 than their native counterparts. GM-CSF–IgG complexes did not bind to cellular GM-CSF receptors, but Fc-dependent binding occurred to blood polymorphonuclear cells. Increased binding of GM-CSF to patient sera correlated positively with the binding capacities of infused IgG preparations. Patient and normal sera did not interfere with the binding of Ab to GM-CSF. From these and previous experiments, we conclude that pools of normal human IgG contain variable amounts of specific and high-avidity Ab to some cytokines, and that Ab to GM-CSF constitute a dominant anti-cytokine activity in these preparations. These Ab are available for reactionin vivo following IgG therapy.

Antibody to Granulocyte-Macrophage Colony-Stimulating Factor Is a Dominant Anti-Cytokine Activity in Human IgG Preparations

Pharmaceutical preparations of normal human immunoglobulin (IgG) are known to contain high-avidity and neutralizing antibodies (Ab) to the cytokines interleukin (IL)-1α, IL-6, and interferon (IFN)α. To test for other cytokine Ab, 23 batches of IgG were tested for saturable binding to eight 125I-labeled recombinant cytokines. All batches bound granulocyte-macrophage colony-stimulating factor (GM-CSF) with high avidity (Kav ≈ 10 pmol/L) and capacities of up to 5 μmol GM-CSF/mol IgG. Only 1 of 15 batches bound IL-5, also with high avidity, whereas 13 of 15 batches bound to IL-10 but with lower capacities and avidities. None of the IgG preparations bound IL-1 receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, or G-CSF. Cross-binding and absorption analyses revealed identical or slightly stronger binding of recombinant GM-CSF, IL-5, and IL-10 than their native counterparts. GM-CSF–IgG complexes did not bind to cellular GM-CSF receptors, but Fc-dependent binding occurred to blood polymorphonuclear cells. Increased binding of GM-CSF to patient sera correlated positively with the binding capacities of infused IgG preparations. Patient and normal sera did not interfere with the binding of Ab to GM-CSF. From these and previous experiments, we conclude that pools of normal human IgG contain variable amounts of specific and high-avidity Ab to some cytokines, and that Ab to GM-CSF constitute a dominant anti-cytokine activity in these preparations. These Ab are available for reactionin vivo following IgG therapy.

Intravenous immunoglobulin preparation increases myoplasmic calcium concentration by activating the dihydropyridine–ryanodine receptor complex

A human intravenous immunoglobulin preparation (IVIg) released Ca 2+ from the sarcoplasmic reticulum of cultured human skeletal muscle cells in a dose-dependent manner. Blocking the dihydropyridine–ryanodine receptor complex abrogated the IVIg-mediated Ca 2+ response, whereas inhibition of the voltage-operated Na +-channels or acetylcholine receptors did not. This effect of IVIg was not mediated by its main component, the IgG molecules, and differed between preparations from different manufacturers. Heating destroyed the activity. Data shows that an unidentified serum protein present in IVIg can influence human muscle cells by an effect on the dihydropyridine receptor. This phenomenon may be important in interpreting the (side) effects of IVIg in neuromuscular diseases.

Real time observation of binding of herpes simplex virus type 1 (HSV-1) to Vero cells and neutralization of HSV-1 by sulfonated human immunoglobulin.

A real time biomolecular interaction assay system involving an optical sensor was applied to the quantitative analysis of the binding of herpes simplex virus type 1 (HSV-1) to Vero cells, and the neutralization antibody titer against this virus with a commercially available sulfonated human immunoglobulin preparation. The virus titers in a viral solution and the neutralizing antibody titer in the human immunoglobulin preparation could be successfully estimated in a short time with this system without any difficult cytopathic effect analysis of cultured cells.

In vitro inhibitory activity of tumor necrosis factor alpha and interleukin-2 of human immunoglobulin preparations.

Human immunoglobulin preparations have been used in a number of clinical settings with good results, although in many of them the mechanism of action is not yet known. One possible mechanism is the modulation of cytokine activity. This study investigated the presence of inhibitory activity in intravenous immunoglobulin (IVIg) and F(ab')2 fragment preparations to two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2). Cytotoxic activity of human recombinant TNF-alpha or TNF-alpha secreted by peripheral blood mononuclear cells (PBMC) on L929 cells and the proliferative activity of the IL-2 on CTLL-2 cells were examined. Human serum albumin (HSA) was used as control. F(ab')2 inhibited, in a dose-dependent fashion, the TNF-alpha activity secreted by PBMC serial dilutions or, at the higher concentrations (25 and 10 mg/ml), recombinant TNF-alpha activity. In contrast, IVIg was able to inhibit only at 25 and 10 mg/ml the TNF-alpha activity secreted by any PBMC dilution tested, and did not inhibit the recombinant TNF-alpha activity. With IL-2, however, even HSA was able to inhibit its proliferative activity, possibly through a carrier effect. The IVIg inhibition of IL-2 activity was not different from that of HSA, but F(ab')2, at 12.5 mg/ml, was capable of inhibiting significantly more the IL-2 activity than HSA. Our results suggest an anticytokine effect of the immunoglobulin preparations that this activity may be mainly mediated by variable regions of the immunoglobulins, and that the more pronounced effect of F(ab')2 may be due to its greater molar concentration compared to intact IgG molecules.

Anti-bacterial activities of a sulfonated human immunoglobulin preparation against penicillin resistant Streptococcus pneumoniae and Pseudomonas aeruginosa

Anti-bacterial activities of a sulfonated human immunoglobulin preparation against penicillin resistant Streptococcus pneumoniae and Pseudomonas aeruginosa were examined. Five week old CBA/J mice were challenged by 10 times of 50% lethal doses of penicillin sensitive (SP1) and resistant (SP2) strains of Streptococcus pneumniae serotype 19, and were treated with a sulfonated human immunoglobulin preparation (hIg). Fifty % protective dose (ED50) were 2-4 mg hIg/ mouse. These doses were parallel with the challenged doses despite of the challenged bacteria, and it was calculated that 6 x 10(6) bacteria were killed by one mg of hIg in both bacteria. Minimal inhibitory concentrations (MIC) of piperacillin, flomoxef, imipenem, amikacin and ofloxacin against SP1 and SP2 were estimated under the presence of various concentrations of hIg. It was found that under the presence of over 10-20 mg/ml of hIg, SP1 and SP2 were not grown even without any antibiotic. That is, MIC of hIg itself against penicillin sensitive and resistant Streptococcus pneumoniae serotype 19 were 10-20 mg/ml. Since about 10(6) bacteria/ml were used for this test, it was calculated that 5-10 x 10(4) bacteria were killed by one mg of hIg in vitro. This result suggested that in vivo anti-bacterial activities of hIg could be 100 times higher than that in vitro. Synergistic or at least additive effects between hIg and all antibiotics tested were seen by the MIC. If hIg was 100 times effective in in vivo, these results suggested that hIg could improve the effect of chemotherapy in clinical cases. Similar in vitro test were carried out for Pseudomonas aeruginosa, and synergistic or at least additive effects between hIg and all antibiotics tested were also confirmed in Pseudomonas aeruginosa. This result suggested that hIg could be effective for clinical pseudomonas infection.

Real time observation of the binding of herpes simplex virus type 1 (HSV-1) to immobilized heparan sulfate and the neutralization of HSV-1 by sulfonated human immunoglobulin.

A real time biomolecular interaction assay system involving an optical sensor was applied to quantitative analysis of the binding of herpes simplex virus type 1 (HSV-1) to immobilized heparan sulfate, a cellular receptor component of HSV-1, and the neutralization antibody titer against this virus with a commercially available sulfonated human immunoglobulin preparation. The virus titer in a viral solution and the neutralizing antibody titer in the human immunoglobulin preparation could be successfully estimated in a short time with this system without any difficult cell culture.

Therapeutic application of intravenous human natural immunoglobulin preparation.

Human natural immunoglobulin (HunIg) preparation for intravenous use has been used in various diseases. The most typical application of this preparation is agammaglobulinemia. Currently, however, this preparation is being used in the therapy of many other disorders. These include thrombocytopenia, Kawasaki disease, systemic vasculitis, several other disorders of autoimmune origin and systemic inflammation such as sepsis. In some diseases, the clinical improvement following use of HunIg has been dramatic, while in others its effect is not striking. Due to rarity of the side effects, the range of application of HunIg has been recently broadened. Such side effects include transmission of several diseases such as hepatitis and retroviral infections. Before it is recommended for use, however, and primarily due to expense, the efficacy of this drug should be carefully evaluated. The mechanism of action of HunIg is not fully understood. However, it has been suggested that its action may involve blockade of Fc-receptor, an anti-cytokine effect, or inhibition of complement activation. In this review, the mechanism of action of HunIgG and its application in human diseases are discussed.

Topical Immunoglobulin Is an Effective Therapy for Parainfluenza Type 3 in a Cotton Rat Model

A cotton rat model was used to test the efficacy of topical immunotherapy against parainfluenza type 3 (PIV3) infection. On day 3 after experimental infection with 10(5.5) pfu of PIV3, animals were treated with 2-fold dilutions of convalescent cotton rat serum or with one of two purified human immunoglobulin preparations; all three had moderate titers of anti-PIV3 neutralizing antibody (range, 1:200-1:1000). Therapy with high concentrations of all three preparations resulted in significant reductions of > or = 2 logs (> or = 100-fold) of pulmonary virus titers compared with titers for control animals. Little or no reduction in virus titers were seen in nasal tissues.

Promotion of remyelination by polyclonal immunoglobulin in Theiler's virus-induced demyelination and in multiple sclerosis.

Spontaneous remyelination occurs in experimental models of demyelination and in patients with multiple sclerosis, although to a limited extent. This enables the search for factors that promote remyelination. Using the Theiler's virus model of central nervous system demyelination, promotion of remyelination was observed after passive transfer of CNS-specific antiserum and transfer of CNS-specific purified immunoglobulin. Mouse polyclonal immunoglobulin from normal non-syngeneic mice, comparable with the human immunoglobulin preparation, also promotes CNS remyelination in the Theiler's virus model of multiple sclerosis. These experimental findings further bridge the gap with a pilot study that suggests clinical improvement after polyclonal immunoglobulin administration, possibly due to remyelination, in some multiple sclerosis patients with stable, steroid-unresponsive optic neuritis. In view of these experimental and clinical data, the physiological role of myelin in demyelination and remyelination is discussed, and the role of IgG solely as a deleterious molecule in the pathophysiology of multiple sclerosis and experimental demyelination is addressed.

Structural modifications of monoclonal antibodies following direct versus indirect labelling with 99Tcm

In this study, the influence of direct and indirect 99Tcm-labelling on the molecular structural integrity of monoclonal antibodies and other immunoglobulin preparations was investigated. Molecular composition of antibody preparations [two IgG monoclonal antibodies, one F(ab')2 fragment (all directly labelled), one indirectly labelled polyclonal human immunoglobulin preparation] and of serum samples after antibody injection were studied using polyacrylamide gel electrophoresis (PAGE; non-reducing and reducing conditions) and gel filtration chromatography. With PAGE, depending on the conditions used, a variety of lower molecular weight products could be detected. When analysing the same antibody preparations by gel filtration chromatography, all complete antibody preparations appeared as homogenous proteins of IgG molecular weight (150 kD). In F(ab')2 fragments, some further fragmentation to Fab' was noticed. Neither in vitro nor in vivo (serum) evidence of smaller fragments could be detected by gel filtration, despite their presence in PAGE. We therefore conclude that through the reductive step of direct 99Tcm-labelling, interchain disulphide linkages are broken but the polypeptide chains of complete IgG remain associated by non-covalent linkages, whereas (F(ab')2 is fragmented further to form essentially Fab'. The protein-denaturating conditions of PAGE (even if performed non-reducingly) seem to produce artifacts, not representing the real in vivo condition. PAGE results should therefore be interpreted only with great care.

Human antibody response to the B oligomer of pertussis toxin.

To determine whether antibodies to the B oligomer of pertussis toxin (PT) were present in patients diagnosed with pertussis or vaccinees who had received diphtheria-tetanus-whole-cell pertussis vaccine, we analyzed serum samples from 5 patients and 10 vaccinees by both enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting techniques. Antibodies to the B oligomer were detected by ELISA in all samples containing antibodies to holotoxin. Western immunoblotting procedures were less efficient than ELISA techniques for detecting antibodies to the B oligomer. Antibodies which inhibit the ability of the B oligomer to agglutinate erythrocytes were detected in purified human immunoglobulin preparations. In addition, serum samples containing antibodies to PT inhibited the binding of purified B oligomer and holotoxin to a 165-kDa glycoprotein which has been considered a potential PT receptor in Chinese hamster ovary (CHO) cells. These results suggest that antibodies to the B oligomer contribute to the human serologic response to PT, but their detection and characterization require appropriate methods.

Inactivation and elimination of viruses during preparation of human intravenous immunoglobulin.

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.

Treatment Of Human Immunodeficiency Virus-Infected Lymphocytes With Cationized Human Immunoglobulins

Hyperimmune immunoglobulins from human immunodeficiency virus (HIV)-infected persons may inhibit intracellular viral replication if the HIV immunoglobulins (HIVIG) are delivered to intracellular spaces where the virus replicates. This study examines the hypothesis that cationized forms of HIVIG undergo enhanced absorptive-mediated endocytosis into cells and inhibit HIV replication. HIVIG and nonimmune human intravenous immunoglobulin (IVIG) were cationized with hexamethylenediamine to an isoelectric point (pI) > 9.5. Cationization markedly increased the binding and endocytosis of HIVIG and IVIG by human peripheral blood lymphocytes (PBL). Cationized HIVIG and IVIG (250 micrograms/mL) resulted in a 90% inhibition in p24 released to the medium in HIV-1-infected human PBL. The study demonstrates that cationization of human immunoglobulin preparations, such as HIVIG and IVIG, markedly increases the endocytosis of immunoglobulin and the inhibition of HIV-1 replication in human PBL.