Anti-monkeypox virus-neutralizing activities of human immunoglobulin manufactured between 1999 and 2021 and derived from donors in the United States and Japan.

BACKGROUND AND OBJECTIVES: In May 2022, the United Kingdom reported the first case of chained transmission of the monkeypox (mpox) virus without any known epidemiological links to west or central Africa. The monthly number of mpox patients currently has passed a peak and is declining globally, and infected patients include both non-vaccinated and vaccinated individuals. Herein, the virus-neutralizing (VN) activity against vaccinia viruses, which are considered to cross-react with the mpox virus, in the intravenous immunoglobulin (IVIG) lots derived from donors, including vaccinated Japanese populations, was evaluated to clarify the status of the Japanese blood donor population. MATERIALS AND METHODS: VN titres against vaccinia and human mpox viruses in IVIG lots derived from donors in Japan and the United States manufactured between 1999 and 2021 and 1995 and 2001, respectively, were evaluated by neutralization testing. RESULTS: VN titres of IVIG derived from donors in Japan and the United States against vaccinia and mpox viruses showed a slowly decreasing trend between 1999 and 2021. CONCLUSION: VN titres are expected to decrease in the future since the percentage of vaccinated donors in the donor population seems to have decreased. Therefore, continuous monitoring of VN titres is required.

Changes in Anti-SARS-CoV-2 Antibody Titers of Pooled Plasma Derived From Donors in Japan: A Potential Tool for Mass-Immunity Evaluation.

The anti-spike (S), anti-nucleocapsid (N), and neutralizing activities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of pooled plasma derived from donors in Japan from January 2021 to April 2022 were evaluated. Anti-S titers and neutralizing activities showed a wave-like trend affected by daily vaccinations and/or the number of reported cases of SARS-CoV-2 infections, whereas anti-N titers remained at negative levels. These results suggest that anti-S and neutralizing titers would fluctuate in pooled plasma in the future. Pooled plasma may be potentially used for mass-immunity evaluation, and titer estimation in intravenous immunoglobulin, a derivative of pooled plasma.

Decline in intravenous immunoglobulin titer against influenza virus A/H2N2 derived from donors in Japan.

Human influenza A/H2N2 can induce a pandemic in the future. This study evaluated the hemagglutination inhibition and neutralizing titers of intravenous immunoglobulin against A/H2N2 viruses, indicating the status of the donor population. In this study, the antibody titers decreased during the study period-2012-2021-suggesting a reduction in the immunity of the studied population.

Long-term monitoring of antibody titers against microorganisms in human intravenous immunoglobulin lots derived from healthy donors in Japan manufactured during 1998-2018.

BACKGROUND: Studies evaluating long-term trends of intravenous immunoglobulin (IVIG) titers against microorganisms such as bacteria and fungi have not been conducted. We herein evaluated long-term trends of titers of IVIG lots manufactured from 1998 to 2018, derived from donors in Japan, against microorganisms affecting patients with primary or secondary immunodeficiency diseases. STUDY DESIGN AND METHODS: Titers against four strains of Pseudomonas aeruginosa, three strains of Staphylococcus aureus, four strains of Haemophilus influenzae, two strains of Klebsiella pneumoniae, one strain of Streptococcus pneumoniae, Enterococcus faecium, Bordetella pertussis, Serratia marcescens, and Candida albicans, were tested immediately after lot release in a commercial clinical testing facility in 1998. The long-term (whole period) and short-term (first and second half of the period) trends of titers were evaluated using regression analysis. RESULTS: The IVIG lots indicated meaningful titers against all microorganisms that were stable in the short term. The long-term trends could be categorized into stable, slightly decreasing, or decreasing trends, except for the metallo-ß-lactamase-producing K. pneumoniae, which indicated a slight increase. Notably, three strains of P. aeruginosa showed remarkable decreasing long-term trends in the titer. DISCUSSION: Some titers indicate decreasing trends against microorganisms over the long-term, however, is not clear whether the phenomenon diminishes the performance of IVIG. The titers of the IVIG lots could provide helpful information to optimize replacement therapy, such as considering trough values based on the titers in the patient plasma. Therefore, continuous monitoring of IVIG titers against microorganisms is important to understand titer fluctuation and epidemiological background.

Screening for parvovirus B19 antigen through chemiluminescent enzyme immunoassay is equivalent to B19 nucleic acid amplification test-based screening of pooled plasma.

BACKGROUND: Human parvovirus B19 (B19) is a pathogen that threatens the quality of plasma products. Therefore, health authorities have mandated measures against B19 contamination of plasma pools. The US FDA has recommended a B19 genome level of 104 IU/ml or lower in pooled plasma lots. Therefore, the B19 nucleic acid amplification test (B19-NAT) has been introduced in many plasma fractionators. However, in the Japanese Red Cross, which is the only approved blood collector in Japan, the B19 antigen test has been introduced for screening donated blood in Japan. Therefore, to clarify whether the antigen test is robust enough to screen blood samples according to the FDA recommendation, we evaluated B19 genome levels in each pooled plasma lot from 2003 to 2020. STUDY DESIGN AND METHODS: Data of 5576 pooled plasma lots from factories A and B, which were derived from plasma bags and passed the B19 antigen-based tests, receptor-mediated hemagglutination assay (B19-RHA), or chemiluminescent enzyme immunoassay (B19-CLEIA), during 2003 to 2020, were evaluated. The amount of B19 genome in each lot was determined using quantitative or semiquantitative B19-NAT. RESULTS: The B19 genome levels in pooled plasma lots screened using B19-RHA did not meet the FDA recommendation, whereas the lots derived from B19-CLEIA fulfilled the FDA recommendation, even during the B19 epidemic in Japan. DISCUSSION: The results suggest that the B19-CLEIA donor screening for plasma pools is also useful in light of the US FDA recommendation.

Neutralizing and binding activities against SARS-CoV-1/2, MERS-CoV, and human coronaviruses 229E and OC43 by normal human intravenous immunoglobulin derived from healthy donors in Japan.

BACKGROUND: There are several types of coronaviruses that infect humans and cause disease. The latest is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is an emerging global threat with no current effective treatment. Normal intravenous immunoglobulin (N-IVIG) has been administered to coronavirus disease 2019 (COVID-19) patients to control severe inflammation and the cellular immune response. However, the neutralizing activity of N-IVIG against SARS-CoV-2 has not yet been fully evaluated. The aim of this study was to determine whether N-IVIG manufactured before the start of the COVID-19 pandemic contained IgG antibodies against the circulating human coronaviruses (HCoVs) that cross-react with the highly pathogenic coronaviruses SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. No cases of SARS-CoV-1 or MERS-CoV have been reported in Japan. STUDY DESIGN AND METHODS: The neutralizing and binding activities of N-IVIG against SARS-CoV-1, MERS-CoV, SARS-CoV-2, HCoV 229E, and HCoV OC43 were evaluated. Nine N-IVIG lots manufactured between 2000 and 2018, derived from donors in Japan, were tested. Binding activity was evaluated by indirect immunofluorescence assay. RESULTS: None of the N-IVIG lots tested displayed neutralizing or binding activity against SARS-CoV-1, MERS-CoV, or SARS-CoV-2. However, they displayed substantial neutralizing and binding activity against HCoV OC43 and weak neutralizing and substantial binding activity against HCoV 229E. CONCLUSION: N-IVIG derived from healthy donors in Japan before the start of the COVID-19 pandemic had no direct effect against SARS-CoV-2. Further studies are warranted to determine the effects of N-IVIG manufactured after the start of the COVID-19 pandemic against SARS-CoV-2.

Long-term monitoring of virus antibody titers in human intravenous immunoglobulin lots derived from donors in Japan.

BACKGROUND: Intravenous immunoglobulin (IVIG) contains immunoglobulin G against various viruses, except those that have been screened, such as human immunodeficiency and hepatitis C viruses. Antivirus titers reflect the serostatus of the blood donor population in the collection region and are of clinical interest. STUDY DESIGN AND METHODS: During the past 10 years, measles, mumps, rubella, varicella-zoster, hepatitis A and B, Epstein-Barr, and human respiratory syncytial viruses; human parainfluenza viruses 1, 2, and 3; human herpes simplex viruses 1 and 2; human herpesvirus 6; cytomegalovirus (CMV); human adenoviruses (HAdVs) 1, 2, 3, 7, and 11; human parvovirus B19; and human echovirus 9 and 11 titers in IVIG lots have been measured by a commercial testing facility. A viral neutralizing assay for CMV has been used at our facility. Herein, we summarize the measurements and results of a regression analysis of the trends in virus antibody titers. RESULTS: IVIG lots contained significant titers against all of the above viruses, except for HAdV 7. Three patterns-stable, increasing, and decreasing-were observed, without any drastic changes. Although these trends reflect the seroprevalence in Japan, the titers were not obviously affected by the cycle of epidemics. On the other hand, the prevalence data suggest that titers against hepatitis A virus and other viruses will decrease in the near future, although they are currently stable. CONCLUSION: Monitoring the titer of IVIG lots and seroprevalence of donor populations is important for anticipating future changes in virus antibody titers of IVIG lots and can provide useful information of clinical interest.

Successful removal of porcine circovirus-1 from immunoglobulin G formulated in glycine solution using nanofiltration.

Porcine circovirus (PCV) is a potentially harmful virus that has been shown to contaminate biological products. The virus is resistant to many inactivation and/or removal procedures performed during manufacturing. Anion exchange chromatography has been shown to be useful for PCV type 1 (PCV1) removal; however, reduction of PCV1 using methods such as heat inactivation, low pH, nanofiltration, UV-C, and gamma irradiation has not been successful. Therefore, in this study, we evaluate various conditions for process solutions during nanofiltration using PCV1. The results indicated that PCV could be effectively removed from glycine solution at 0.1-0.3 M, pH 4.0 without IgG, using a nanofilter with a pore size of 19 nm (19-nm filter); log reduction values (LRVs) of ≥4.5 and ≥ 5.0, respectively, were obtained. In contrast, PCV1 was significantly removed (LRV: 2.2) in glycine solution at 0.3 M, pH 6.0 with 1.0% IgG using the 19-nm filter, but some virus genomes were detected in the filtrates. In summary, the use of a 19-nm filter in glycine solution with/without IgG is an appropriate condition for PCV removal.

Hepatitis E virus derived from different sources exhibits different behaviour in virus inactivation and/or removal studies with plasma derivatives.

Hepatitis E virus (HEV) causes viral hepatitis, and is considered a risk factor for blood products. Although some HEV inactivation/removal studies have been reported, detailed investigations of different manufacturing steps as heat treatment, partitioning during cold ethanol fractionation, low pH treatment, and virus filtration have yet to be reported for plasma-derived medicinal products. In this study, human serum- and swine faeces-derived HEVs, with and without detergent treatment, were used. The kinetic patterns of inactivation, log reduction value, or partitioning during the process were evaluated. In addition, the mouse encephalomyocarditis virus (EMCV) and canine and porcine parvoviruses (CPV/PPV) were also evaluated as model viruses for HEV. Small pore size (19 or 15 nm) virus filtration demonstrated effective removal of HEV. Middle pore size (35 nm) virus filtration and 60 °C liquid heating demonstrated moderate inactivation/removal. Ethanol fractionation steps demonstrated limited removal of HEV. Unpurified HEV exhibited different properties than the detergent-treated HEV, and both forms displayed differences when compared with EMCV, CPV, and PPV. Limited or no inactivation of HEV was observed during low pH treatment. Untreated plasma-derived HEV from humans showed different properties compared to that of HEV treated with detergent or derived from swine faeces. Therefore, HEV spike preparation requires more attention.

Virus Neutralization by Human Intravenous Immunoglobulin Against Influenza Virus Subtypes A/H5 and A/H7.

PURPOSE: Highly pathogenic avian influenza viruses are a threat to human health. Although donor populations have not experienced pandemic, they have been immunized by natural infections and/or vaccinations of influenza viruses such as A/H1N1, A/H3N2, and B. Therefore, it is considered that human intravenous immunoglobulin (IVIG) derived from healthy donors does not include IgG against avian influenza viruses. However, cross-reactivity has not been evaluated yet. In this study, cross-reactivity against the avian influenza virus A/H5N1, A/H7N1, A/H7N2, A/H7N7, A/H7N9, and A/H10N9 was evaluated. MATERIALS AND METHODS: Several lots of IVIG derived from healthy donors in Japan were tested for virus neutralization using single- or multi-cycle virus neutralizing (S-VN or M-VN) assays that evaluate the infection-step associated with HA or the infection and propagation steps associated with HA and NA, respectively. In addition, anti-NA activities were evaluated by inhibiting the enzymatic activity in NAI assays. RESULTS: IVIG lots showed high neutralizing activities against three A/H5N1 strains in M-VN assays, whereas activities in S-VN assays were unstable. In addition, A/H7N2 was also neutralized in S-VN and M-VN assays, with higher activity in M-VN than in S-VN assays. A/H7N1 was neutralized in S-VN and M-VN assays. In contrast, weak or no activity against A/H7N7, A/H7N9, and A/H10N9 was observed in S-VN and M-VN assays. NAI assay results show that IVIG lots had inhibitory activities against N1 and N2; however, N2 activities differed depending on the strain. In contrast, no activities were observed against N7 and N9. CONCLUSION: These results suggest that IVIG lots have neutralizing activity against avian influenza viruses during the virus propagation step, except for one strain, although no or weak activity was observed during the infection step.

Polyethyleneimine-modified resins effectively remove porcine circovirus and cellular prion protein.

Polyethyleneimine (PEI) possesses various molecular weights (MWs), structures, and virus capture capacities. However, whether PEI can capture porcine circovirus (PCV) and animal cell-derived prion protein (PrPC) that may contaminate source materials is unclear. Therefore, we conducted a feasibility study to assess the effectiveness of PEI in removing PCV and PrPC as a model of pathogenic prions. The removal performance of PCV was evaluated by quantitative PCR using PEIs with various MWs, structures, and ion exchange capacities in Tris (pH 7.5) and acetate (pH 5.5) buffers under neutral (pH 7.5) to acidic (pH 5.5) conditions. Removal performances of PrPC were also evaluated by western blotting using PEIs with various MWs and structures. Tris buffer did not affect the ability of PEI-modified resins to remove PCV, whereas acetate buffer affected removal performances, except those of PEI-10K-Br and PEI-70K-Br, which showed high ion-exchange capacities. PrPC was captured by PEIs with high MWs, especially PEI-70K-Br, which was the most effective. The results of this feasibility study suggested that PEI-modified resin could remove PCV and PrPC. PEI-70K-Br with an ion-exchange capacity of at least 0.3 meq/mL appears suitable as a PEI molecule for pathogen capture or removal of PCV or PrPC from biological materials.

Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition.

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.

Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies.

Pigs are presumed reservoirs for hepatitis E virus (HEV) transmission to humans. To examine infection kinetics, two litters of domestic pigs (A and B, each containing 10 piglets) infected naturally with HEV were studied until pigs were 6 months old. Maternal IgG and IgA antibodies were detected in litter A piglets, but not in litter B ones. All pigs shed HEV in feces when they were 30-110 days old, and 17 developed viremia at 40-100 days of age. Phylogenetic analysis revealed a highly close sequence of HEV genotype 3 in all pigs. The serum levels of specific IgG and IgA were similar in all pigs, although IgA was not detected in the feces. Interestingly, the onset of both viremia and seroconversion was delayed significantly in litter A pigs. The kinetics of fecal virus shedding was similar in both litters; shedding was not detected after the pigs were 120 days old. The differences in the infection kinetics between litters A and B suggested that maternal antibodies delayed the onset of viremia and seroconversion. Quantitative real-time reverse transcriptase-polymerase chain reaction revealed that HEV RNA in feces peaked 10 days after initial shedding of approximately 10(6.0) copies/g. The viral load was much lower in the serum than in the feces. At 200 days of age, HEV RNA was found in the internal organs of 3 out of 13 pigs. These study findings improve the understanding of the dynamics of natural HEV transmission in pigs, which could help in controlling virus transmission from pigs to humans.

Molecular detection of genotype 3 porcine hepatitis E virus in aborted fetuses and their sows.

By reverse transcription (RT)-nested polymerase chain reaction (PCR), hepatitis E virus (HEV) RNA was simultaneously detected in the livers of aborted fetuses and in fecal and serum samples from their sows, obtained from two of 14 farms tested. Sequence and phylogenetic analyses showed that these HEVs belonged to genotype 3 HEV. HEV RNA was detected by real-time RT-PCR at 2.0 x 10(3)-2.8 x 10(4) viral copies/microl in the HEV-positive samples. All HEV-positive fetuses tested positive for porcine circovirus type 2 (PCV2). These results indicate that transplacental infection of HEV with PCV2 coinfection may occur in sows with reproductive failure, which is suggestive of similarities to HEV infection in women.

Infectious prion protein in the filtrate even after 15 nm filtration.

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was >or=4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than approximately 15 nm in diameter).

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors.

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

Neutralizing activities against seasonal influenza viruses in human intravenous immunoglobulin.

Influenza viruses A/H1N1, A/H3N2, and B are known seasonal viruses that undergo annual mutation. Intravenous immunoglobulin (IVIG) contains anti-seasonal influenza virus globulins. Although the virus-neutralizing (VN) titer is an indicator of protective antibodies, changes in this titer over extended time periods have yet to be examined. In this study, variations in hemagglutination inhibition (HI) and VN titers against seasonal influenza viruses in IVIG lots over extended time periods were examined. In addition, the importance of monitoring the reactivity of IVIG against seasonal influenza viruses with varying antigenicity was evaluated. A/H1N1, A/H3N2, and B influenza virus strains and IVIG lots manufactured from 1999 to 2014 were examined. The HI titer was measured by standard methods. The VN titer was measured using a micro-focus method. IVIG exhibited significant HI and VN titers against all investigated strains. Our results suggest that the donor population maintains both specific and cross-reactive antibodies against seasonal influenza viruses, except in cases of pandemic viruses, despite major antigen changes. The titers against seasonal influenza vaccine strains, including past strains, were stable over short time periods but increased slowly over time.