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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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‐β‐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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatitis E virus (HEV) transmitted via the oral route through the consumption of contaminated water or uncooked or undercooked contaminated meat has been implicated in major outbreaks. Rats may play a critical role in HEV outbreaks, considering their negative effects on environmental hygiene and food sanitation. Although the serological evidence of HEV infection in wild rodents has been reported worldwide, the infectivity and propagation of HEV in wild rats remain unknown. To investigate if rats are a possible carrier of HEV, we studied wild Norway rats (Rattus norvegicus) that were caught near a pig farm, where HEV was prevalent among the pigs. Methods We examined 56 Norway rats for HEV. RNA from internal organs was examined for RT-PCR and positive samples were sequenced. Positive tissue samples were incubated with A549 cell line to isolate HEV. Anti-HEV antibodies were detected by ELISA. Results Sixteen rats were seropositive, and the HEV RNA was detected in 10 of the 56 rats. Sequencing of the partial ORF1 gene from 7 samples resulted in partially sequenced HEV, belonging to genotype 3, which was genetically identical to the HEV prevalent in the swine from the source farm. The infectious HEVs were isolated from the Norway rats by using the human A549 cell line. Conclusions There was a relatively high prevalence (17.9%) of the HEV genome in wild Norway rats. The virus was mainly detected in the liver and spleen. The results indicate that these animals might be possible carrier of swine HEV in endemic regions. The HEV contamination risk due to rats needs to be examined in human habitats.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Manufacturing processes used in the production of biopharmaceutical or biological products should be evaluated for their ability to remove potential contaminants, including TSE agents. In the present study, we have evaluated scrapie prion protein (PrPSc) removal in the presence of different starting materials, using virus removal filters of different pore sizes. Following 75nm filtration, PrPSc was detected in the filtrate by Western blot (WB) analysis when a “super-sonicated” microsomal fraction derived from hamster adapted scrapie strain 263K (263K MF) was used as the spike material. In contrast, no PrPSc was detected when an untreated 263K MF was used. By using spike materials prepared in a manner designed to optimize the particle size distribution within the preparation, only 15nm filtration was shown to remove PrPSc to below the limits of detection of the WB assays used under all the experimental conditions. However, infectious PrPSc was recovered following 15nm filtration under one experimental condition. The results obtained suggest that the nature of the spike preparation is an important factor in evaluating the ability of filters to remove prions, and that procedures designed to minimize the particle size distribution of the prion spike, such as the “super-sonication” or detergent treatments described herein, should be used for the preparation of the spike materials. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Influenza A H2N2 virus, also known as the Asian flu, spread worldwide from 1957 to 1967, although there have been no cases reported in humans in the past 40 years. A vaccination program was introduced in Japan in the 1960s. Older Japanese donors could have been naturally infected with the H2N2 virus or vaccinated in the early 1960s. Human intravenous immunoglobulin (IVIG) reflects the epidemiological status of the donating population in a given time period. Here, the possible viral neutralizing (VN) activities of IVIG against the H2N2 virus were examined. Hemagglutination inhibition (HI) and VN activities of IVIG lots manufactured from 1993 to 2010 in Japan and the United States were evaluated against H2N2 viruses. High HI and VN activities against H2N2 viruses were found in all the IVIG lots investigated. HI titers were 32-64 against the isolate in 1957 and 64-128 against the isolates in 1965. VN titers were 80-320 against the isolate in 1957 and 1280-5120 against the isolates in 1965. Both the HI and VN titers were higher against the isolate in 1965 than in 1957. Thus, antibody titers of IVIG against influenza viruses are well correlated with the history of infection and the vaccine program in Japan. Therefore, evaluation of antibody titers provides valuable information about IVIGs, which could be used for immune stimulation when a new influenza virus emerges in the human population. [ABSTRACT FROM AUTHOR]

Abstract: 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 15nm 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 ≥4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15nm 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 ∼15nm in diameter). [Copyright &y& Elsevier]

Abstract: 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 β-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 β-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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus. [Copyright &y& Elsevier]

Abstract: The potential contamination of human blood or plasma with prions, such as variant Creuftzfelt-Jacob disease (vCJD), is becoming a serious problem. In this study, we established a Western blot-based detection method for PrPSc (263K) spiked in plasma. Although plasma contains a large amount of protein, specific detection of a small amount of 263K in plasma could be specifically detected only after ultra-centrifugation followed by heat-denaturation of plasma proteins included in the resulting precipitate, before the digestion with proteinase K. [Copyright &y& Elsevier]