Purification of intravenous immunoglobulin G from human plasma--aspects of yield and virus safety

Plasma-derived intravenous immunoglobulin (IVIG) preparations have been successfully applied for the prophylactic prevention of infectious diseases in immunodeficient patients. In addition to its replacement therapy of primary and secondary antibody deficiencies, IVIG has found increased use in autoimmune and inflammatory diseases. IVIG has become the major plasma product on the global blood product market. The world wide consumption nearly tripled between 1992 and 2003, from 19.4 to 52.6 tons. Classical manufacturing processes of IVIG, but also new strategies for purification are discussed with respect to practicability and yield. Ethanol fractionation is still the basis for most IVIG processes, although isolation and purification of immunoglobulin G (IgG) by chromatography has gained ground. The efficiency of virus inactivation methods and virus removal techniques in terms of logarithmic reduction factors are analyzed, but also the IgG losses are taken into consideration. Some of these methods also have the ability to separate prions. High pathogen safety and high yields have become the dominant goals of the plasma fractionation industry.     

False-positive human immunodeficiency virus enzyme immunoassay results in pregnant women

Objective

Examine whether false-positive HIV enzyme immunoassay (EIA) test results occur more frequently among pregnant women than among women who are not pregnant and men (others).

Design

To obtain a large number of pregnant women and others tested for HIV, we identified specimens tested at a national laboratory using Genetic Systems HIV-1/HIV-2 Plus O EIA from July 2007 to June 2008.

Methods

Specimens with EIA repeatedly reactive and Western blot-negative or indeterminate results were considered EIA false-positive. We compared the false-positive rate among uninfected pregnant women and others, adjusting for HIV prevalence. Among all reactive EIAs, we evaluated the proportion of false-positives, positive predictive value (PPV), and Western blot bands among indeterminates, by pregnancy status.

Results

HIV prevalence was 0.06% among 921,438 pregnant women and 1.34% among 1,103,961 others. The false-positive rate was lower for pregnant women than others (0.14% vs. 0.21%, odds ratio 0.65 [95% confidence interval 0.61, 0.70]). Pregnant women with reactive EIAs were more likely than others (p<0.01) to have Western blot-negative (52.9% vs. 9.8%) and indeterminate results (17.0% vs. 3.7%) and lower PPV (30% vs. 87%). The p24 band was detected more often among pregnant women (p<0.01).

Conclusions

False-positive HIV EIA results were rare and occurred less frequently among pregnant women than others. Pregnant women with reactive EIAs were more likely to have negative and indeterminate Western blot results due to lower HIV prevalence and higher p24 reactivity, respectively. Indeterminate results may complicate clinical management during pregnancy. Alternative methods are needed to rule out infection in persons with reactive EIAs from low prevalence populations.     

Epstein-Barr virus serology

Because of the ubiquitous nature of EBV, most people are infected with this virus by the time they are adults. People acquire the virus at an early age, earlier in developing countries and in socioeconomically deprived areas of the United States, where about 80% of 5-year-old children are seropositive. In economically privileged areas, only about 40–50% of children are seropositive by age 5. Infections during childhood are usually asymptomatic. In contrast, 50% of adolescents who become infected with EBV develop the fatigue, fever, pharyngitis, and atypical lymphocytosis characteristic of acute infectious mononucleosis (IM). Heterophil antibodies, which are the basis for screening tests for IM, usually appear in the serum of these patients. However, approximately 10% of patients (more commonly children) with EBV induced IM do not develop heterophil antibodies. For this reason, tests for specific antibody-mediated immune responses to EBV may be necessary for diagnosis.     

Inactivation of human T-lymphotropic virus type III/lymphadenopathy-associated virus by formaldehyde-based reagents

Neutral buffered Formalin, a fixative used in most pathology laboratories, was found to inactivate human T-lymphotropic virus type III/lymphadenopathy-associated virus. Preparations containing this virus with infectivity titers of greater than 10(5) were treated with 1% or greater neutral buffered Formalin; after treatment, virus was undetectable (titer, less than 10(1)). In addition, when infected phytohemagglutinin-stimulated lymphocytes were treated with paraformaldehyde, transmission of the virus to other such lymphocytes was eliminated.     

Inactivation of human T-lymphotropic virus type III/lymphadenopathy-associated virus by formaldehyde-based reagents.

Neutral buffered Formalin, a fixative used in most pathology laboratories, was found to inactivate human T-lymphotropic virus type III/lymphadenopathy-associated virus. Preparations containing this virus with infectivity titers of greater than 10(5) were treated with 1% or greater neutral buffered Formalin; after treatment, virus was undetectable (titer, less than 10(1)). In addition, when infected phytohemagglutinin-stimulated lymphocytes were treated with paraformaldehyde, transmission of the virus to other such lymphocytes was eliminated.     

Complex cell cycle abnormalities caused by human T-lymphotropic virus type 1 Tax

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4(+) T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27(Kip1) and p21(CIP1/WAF1) through protein stabilization and mRNA trans-activation and stabilization, respectively. The Tax-induced surge in p21(CIP1/WAF1) and p27(Kip1) begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and fluorescent ubiquitin cell cycle indicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated β-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency.     

The serology and immunochemistry of HIV-induced platelet-bound immunoglobulin

A study was carried out on the presence of platelet-bound immunoglobulins, platelet-bound complement and serum immunoglobulin reactive with platelets in the blood of persons infected with HIV and those at risk of HIV infection. Platelet-bound immunoglobulins, predominantly IgG and IgM, but not complement, were demonstrated by immunofluorescence in 16 out of 16 patients with AIDS, in 5 out of 7 with AIDS-related complex/persistent generalized lymphadenopathy and in 7 out of 10 apparently healthy sexually active homosexual men, of whom 2 were anti-HIV1 seropositive. There was no correlation between the presence of platelet-bound immunoglobulins and either the platelet count or the level of circulating immune complexes. The specificity of the platelet-bound immunoglobulins and platelet-reactive immunoglobulins in the corresponding sera was studied. Platelet-bound immunoglobulins were eluted and then investigated for cross-reactivity with HIV. Both sera and eluates were tested for reactivity with cardiolipin and reactivity with the major target antigen in classical autoimmune thrombocytopenia, the GP IIb/IIIa complex. Of 17 eluates containing platelet-reactive immunoglobulins, 5 reacted with HIV-determinants but 2 out of 5 eluates that did not contain platelet-reactive immunoglobulins also reacted. Although anti-cardiolipin antibodies were detected in all sera, none of the 17 eluates reacted with cardiolipin. Moreover, sera and eluates, reactive with normal platelets, did not react with type-1-Glanzmann disease platelets. This indicates that the antibodies are directed against the glycoprotein IIb/IIIa complex of platelets. This could not be confirmed by immunoprecipitation or by immunoblotting, however. We conclude that the presence of platelet-bound immunoglobulins is common in HIV-infection but may also occur in persons at risk and that the nature of the auto-antibodies is not different from that of the auto-antibodies observed in classical ITP.     

Isolation of a human T-lymphotropic virus type I strain from Australian aboriginals.

A human T-lymphotropic virus type I (HTLV-I) strain was isolated in a CD4+ T-lymphocyte culture established from a healthy seropositive Australian Aboriginal. This isolate, identified as HTLV-IMSHR-1, was detected by immunofluorescence with monoclonal antibodies, by the presence of gag-encoded protein p24 in the culture supernatant, and by cocultivation leading to infection and transformation of lymphocytes from an HTLV-I-negative donor. By using the polymerase chain reaction technique, the env gene and segments of the pol and pX regions of the proviral genome of HTLV-I(MSHR-1) were amplified and sequenced. Comparison with the envelope sequences of prototype strains revealed up to 7% divergence at the nucleotide level and 3.1 to 4.3% divergence in the predicted amino acid sequence. Phylogenetic analysis showed that the Australian and Melanesian isolates are related. Differential reactivity with monoclonal antibodies suggests that gag protein p19 of HTLV-I(MSHR-1) is also divergent. The potential for antigenic divergence between the prototype HTLV-I isolates and the Austro-Melanesian variants requires further investigation, because it would have implications for serodiagnosis and vaccine development.     

Sporadic cases of carriers of human T-lymphotropic virus type 1 in Southeast Asia

Sera obtained from 3,472 persons in Malaysia, Thailand, Philippines and Indonesia were tested for the presence of antibody to adult T-cell leukemia-associated antigen by the gelatin particle agglutination test and indirect immunofluorescence. Among these, only two seropositives were identified. One was a 30-year-old male Malaysian of Indian origin. The other was a 42-year-old female Thai who resided in Bangkok. These results suggested that the infection of human T-lymphotropic virus type 1 might not be endemic in these countries.     

Infection with the human T-lymphotropic virus type I. A review for clinicians.

The human T-cell lymphotropic virus type I (HTLV-I) is the first retrovirus identified in humans. It has been responsible for a number of clinical syndromes, most notably adult T-cell leukemia or lymphoma and tropical spastic paraparesis. In the United States, infection with this virus is most frequently found in specific subsets of our population, particularly in those who live in the southeastern states, have southern Japanese ancestry, or share intravenous drug paraphernalia. Understanding the epidemiology and clinical manifestations of this virus is necessary to properly diagnose and care for patients with HTLV-I infection.     

Human intravenous immunoglobulin preparation and virus inactivation by pasteurization and solvent detergent treatment

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 degrees C for 10 hours, or by ion exchange chromatography and solvent/detergent treatment. The final preparations, formulated in 5% immunoglobulin solutions were characterized by in vitro analyses of biochemical and biological properties and compared with the samples of other manufacturer's IVIG solution products. The critical properties evaluated in this study were purity, molecular intactness, and the biological functions such as Fc function and anticomplementary activity. Virus inactivation and removal by processing steps and by deliberate virucidal steps, as described above, were tested on various human pathogenic viruses, such as human immunodeficiency and experimental model viruses. The tested viruses were successfully inactivated and removed. We conclude that the intravenous immunoglobulins prepared by two different methods, as described above, provide an equivalent viral safety and quality.     

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface-exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.     

Monocyte-driven activation-induced apoptotic cell death of human T-lymphotropic virus type I-infected T cells.

We attempted apoptotic cell death induction of T cells infected with human T lymphotropic virus type I (HTLV-I) which induces HTLV-I-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. T cells acutely infected and expressing HTLV-Igag Ags were killed by cross-linking their TCR with anti-CD3 mAb. Cells in apoptotic process were found by staining with annexin V. The apoptosis was not affected by costimulation through CD28 molecules and was resistant to ligation of Fas molecules. Whereas the virus-infected T cells expressed higher levels of HLA-DR, CD25, CD80, and CD86 Ags than apoptosis-resistant PHA-blasts, the T cell apoptosis was enhanced by addition of exogenous IL-2. Furthermore, in this apoptosis, monocytes played an important role because T cells infected in the absence of monocytes were resistant to the death signals. The apoptosis-sensitive T cells responded to TCR signaling more strongly by proliferating than those apoptosis-resistant cells. Monocytes weakly affected the expression levels of viral Ags on T cells. However, HTLV-I-infected monocytes primed T cells to die by subsequent TCR signaling. T cells primed with the monocytes, subsequently infected in the absence of monocytes, were killed by TCR signaling. These observations suggest that primed and infected T cells could be killed by activation-induced cell death.     

Serological differences between human T-lymphotropic virus type-I (HTLV-I) and HTLV-III/LAV

Sera from each of five preselected groups of patients with acquired immune deficiency syndrome (AIDS), AIDS-related complex (ARC), hemophilia, adult T-cell leukemia (ATL), and healthy controls were examined for antibodies to human T-cell leukemia (T-lymphotropic) virus type-I (HTLV-I) and HTLV-III by indirect immunofluorescence (IF) and radioimmunoprecipitation (RIP) methods. All sera from five patients with AIDS, ARC, and hemophilia reacted at titers from 1 : 512 to 1 : 5,120 with fixed H9/HTLV-III cells by IF but not with fixed MT-1 cells carrying HTLV-I. Similarly, sera from patients with AIDS, ARC, and hemophilia precipitated HTLV-III-specific polypeptides of 120K, 46K, and 24K. In contrast, sera from five patients with ATL did not react with fixed H9/HTLV-III cells, but reacted with fixed MT-1 cells. Moreover, HTLV-I-specific polypeptides of 68K, 28K, and 24K were precipitated with sera from ATL-patients but not with anti-HTLV-III-positive sera. Recently, we infected HTLV-I-carrying MT-4 cells with HTLV-III and provoked strong cytopathic effects. This system enabled testing for neutralizing antibodies to HTLV-III. Neutralizing titers to HTLV-III of five anti-HTLV-III-positive sera ranged from 1 : 720 to 1 : 9,000. In contrast, all five seronegative controls showed no or only low reactivity to HTLV-III envelope (1 : 80 and 100). However, three out of five anti-HTLV-I-positive sera exhibited weak cross-reactivities with HTLV-III. The reactivities were expressed as less than 1 : 160, except for one case (1 : 720). They were considered to be nonspecific since they were negative for HTLV-III antibodies in the radioimmunoprecipitation studies.