Neutralization of Rubella Vaccine Virus and Immunodeficiency-Related Vaccine-Derived Rubella Viruses by Intravenous Immunoglobulins.

The association between granulomas and vaccine-derived rubella virus (VDRV) in people with primary immune deficiencies (PID) has raised concerns about the ability of immunoglobulin (IG) preparations to neutralize VDRVs. We investigated the capacity of IG to neutralize rubella vaccine virus and four VDRV strains. As expected, the rubella vaccine virus itself was potently neutralized by IG preparations; however, the VDRV isolates from patients after intra-host evolution, 2-6 times less so. Diagnosis of immune deficiencies before possible live-virus vaccination is thus of critical importance, while IG replacement therapy can be expected to provide protection from rubella virus infection.

The occurrence of granulomas associated with vaccine derived rubella viruses (VDRV) in people with primary immune deficiencies (PID) challenges immunoglobulin (IG) preparations regarding their rubella neutralizing ability. This study confirmed potent rubella virus neutralization capacity of IG preparations and thus suggests protection of IG-treated PID patients against rubella. The study also highlights the importance of early diagnosis and timely given IG to prevent possible systemic spread of VDRV persisting locally in granulomas.

Human Circovirus is not detected in plasma pools for fractionation.

BACKGROUND: Human Circovirus 1 and 2 were recently described in a French hepatitis case and in two Chinese drug users. Because of its small size and presumable high resistance to both inactivation and removal by nanofilters, such viruses-if determined to be even pathogenic-should be considered with respect to the safety of plasma derivatives. We, therefore, investigated the prevalence and titer of these viruses in plasma pools before fractionation. METHODS AND MATERIALS: We tested for the presence of Human Circovirus 1 and 2 by qPCR in 48 plasma pools derived from healthy donors from Europe, USA, and Japan, corresponding to more than 200,000 plasma donations. RESULTS: We did not detect the presence of Human Circovirus 1 and 2 in any of the plasma pools, with a limit of detection of 300-600 genome copies per mL of plasma. CONCLUSIONS: These results indicate that high levels of circovirus are not widely prevalent in such donations.

Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work.

On-column virus inactivation by solvent/detergent treatment for a recombinant biological product.

Each process step in the manufacture of biological products requires expensive resources and reduces total process productivity. Since downstream processing of biologicals is the main cost driver, process intensification is a persistent topic during the entire product life cycle. We present here one approach for the intensification of bioprocesses by applying on-column virus inactivation using solvent/detergent (S/D) treatment during ion-exchange chromatography. The established purification process of a recombinant protein was used as a model to compare key process parameters (i.e., product yield, specific activity, impurity clearance) of the novel approach to the standard process protocol. Additional wash and incubation steps with and without S/D-containing buffers were introduced to ensure sufficient contact time to effectively eliminate enveloped viruses and to significantly decrease the amount of S/D reagents. Comparison of key process parameters demonstrated equivalent process performance. To assess the viral clearance capacity of the novel approach, XMuLV was spiked as model virus to the chromatographic load and all resulting fractions were analyzed by TCID50 and RT-qPCR. Data indicates the inactivation capability of on-column virus inactivation even at 10% of the nominal S/D concentration, although the mechanism of viral clearance needs further investigation.

Rapidly Increasing Severe Acute Respiratory Syndrome Coronavirus 2 Neutralization by Intravenous Immunoglobulins Produced From Plasma Collected During the 2020 Pandemic.

Immunoglobulin lots (N = 176) released since March 2020 were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies, with first positive results for September 2020 lots (mean, 1.7 IU/mL; 46% of lots positive). From there, values steadily increased, in correlation with the cumulative coronavirus disease 2019 (COVID-19) incidence, to reach a mean of 31.2 IU/mL and 93% of lots positive by January 2021. Extrapolating the correlation, immunoglobulins could reach an anti-SARS-CoV-2 potency of approximately 345 IU/mL by July 2021. At that stage, prophylactic immunoglobulin treatment for primary/secondary immunodeficiency could contain similar doses of anti-SARS-CoV-2 as convalescent plasma that is used for treatment of COVID-19.

Omicron Severe Acute Respiratory Syndrome Coronavirus 2 Neutralization by Immunoglobulin Preparations Manufactured From Plasma Collected in the United States and Europe.

After >2 years of the coronavirus disease 2019 (COVID-19) pandemic, immunoglobulins (IGs) contain highly potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies, based on the large proportion of United States (US) plasma donors who have gone through COVID-19 or vaccination against the virus. Neutralization of Omicron SARS-CoV-2 by antibodies generated after non-Omicron infection or vaccination has been lower though, raising concerns about the potency of IG against this new virus variant. Also, as plasma collected in the US remains the main source of IG, the neutralization of SARS-CoV-2 for plasma collected elsewhere has been less well studied. Here, we confirm Omicron neutralization by US as well as European Union plasma-derived IG lots.

Highly Potent SARS-CoV-2 Neutralization by Intravenous Immunoglobulins manufactured from Post-COVID-19 and COVID-19-Vaccinated Plasma Donations.

From September 2020, some immunoglobulin lots from US plasma contained neutralizing antibodies against the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Paralleled by the increasing numbers of post-coronavirus disease 2019 (COVID-19) donors, immunoglobulin lot antibody positivity increased to 93% by January 2021, at a mean titer of approximately 30 IU/mL. The correlation predicted that anti-SARS-CoV-2 potency would reach 345 IU/mL by July 2021. In addition to post-COVID-19 donors, the rapidly increasing number of plasma donors vaccinated against COVID-19 resulted in a mean antibody titer of >600 IU/mL in July 2021 immunoglobulin lots, with SARS-CoV-2 antibody titers for several lots even higher than those of earlier produced hyperimmune globulin products.

Longitudinal analysis of SARS-CoV-2 antibodies in 8000 U.S. first-time convalescent plasma donations.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent individuals carry antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that, through a plasma donation, can be used as a potential therapeutic either in direct transfusion or for the manufacture of hyperimmune globulin (HIG). The success of such interventions depends on the antibody potency in such plasma donations, but little information on the collection of potent units is currently available. STUDY DESIGN AND METHODS: A total of 8749 plasma units, collected from April until September 2020 from first-time U.S. COVID-19 convalescent plasma donors, were characterized for SARS-CoV-2 immunoglobulin G (IgG) antibodies by Abbott chemiluminescent microparticle immunoassay (CMIA). The period between COVID-19 onset until donation and donor age, ethnicity, sex, and COVID-19 severity were evaluated against the obtained signal (index S/C). RESULTS: A marked decrease in mean index S/C was seen over the plasma collection period surveyed, which was significantly correlated to decreases in mean plasma donor age (p < .0001; R2 = .726) and percentage of donations obtained from COVID-19 convalescent patients who had been hospitalized (p = .001; R2 = .4426). The highest titer plasma units were obtained soon after convalescence from COVID-19 patients who required hospitalization, from advanced age donors, and from Black/African/Hispanic American versus White/Caucasian ethnicities, whereas there was no effect of donor sex on the values obtained with the Abbott CMIA. CONCLUSION: Since the onset of the pandemic, the average SARS-CoV-2 IgG values of first-time U.S. COVID-19 convalescent plasma donations have significantly dropped, mainly due to donations from progressively younger aged donors who tend to experience less severe COVID-19.

Characterization of 100 sequential SARS-CoV-2 convalescent plasma donations.

BACKGROUND: Transfusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent plasma is a promising treatment for severe coronavirus disease 2019 (COVID-19) cases, with success of the intervention based on neutralizing antibody content. Measurement by serologic correlates without biocontainment needs as well as an understanding of donor characteristics that may allow for targeting of more potent donors would greatly facilitate effective collection. STUDY DESIGN AND METHODS: One hundred convalescent plasma units were characterized for functionally active SARS-CoV-2 neutralizing antibodies, as well as for SARS-CoV-2 binding antibodies, with the intention to establish a correlation between the functionally more relevant neutralization assay and the more accessible enzyme-linked immunosorbent assay (ELISA). Donor demographics such as COVID-19 severity, age, and sex were correlated with antibody titers. RESULTS: A mean neutralization titer 50% of 230 (range, <8-1765) was seen for the 100 convalescent plasma units, with highly significant (P < .0001) yet quantitatively limited (R2 = 0.2830) correlation with results of the ELISA. Exclusion of units with particularly high titers (>500) from analysis improved correlation (R2 = 0.5386). A tendency of higher-titer plasma units from donors with increased disease severity, of advanced age, and of male sex was seen, yet the functional relevance of this difference is questionable. CONCLUSION: The ELISA-based correlation to neutralization titer enabled a threshold proposal that could be used to eliminate lower-titer units from the clinical supply for COVID-19 treatment. Disease severity may be associated with the development of higher titers of neutralizing antibodies, although larger case numbers will be needed for additional confirmation.

No SARS-CoV-2 Neutralization by Intravenous Immunoglobulins Produced From Plasma Collected Before the 2020 Pandemic.

The 2020 SARS-CoV-2 pandemic is caused by a zoonotic coronavirus transmitted to humans, similar to earlier events. Whether the other, seasonally circulating coronaviruses induce cross-reactive, potentially even cross-neutralizing, antibodies to the new species in humans is unclear. The question is particularly relevant for people with immune deficiencies, as their health depends on treatment with immunoglobulin preparations that need to contain neutralizing antibodies against the pathogens in their environment. Testing 54 intravenous immunoglobulin preparations, produced from plasma collected in Europe and the United States, confirmed highly potent neutralization of a seasonal coronavirus; however, no cross-neutralization of the new SARS-CoV-2 was seen.

Antibody-enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin.

BACKGROUND: Circulation of hepatitis E virus (HEV) in areas where plasma is sourced for the manufacture of plasma-derived medicinal products (PDMPs) has prompted verification of HEV clearance. HEV exists as quasi lipid-enveloped (LE) and non-lipid-enveloped (NLE) forms, which might be of relevance for HEV clearance from manufacturing processes of antibody-containing PDMPs with solvent/detergent (S/D) treatment upstream of further clearance steps. STUDY DESIGN AND METHODS: Presence of different HEV particles in stocks used in clearance studies was investigated, with nanofilters graded around the assumed HEV particle sizes and by gradient centrifugation. HEV removal by 35-nm nanofiltration was investigated in the presence or absence of HEV antibodies, in buffer as well as in immunoglobulin (IG) manufacturing process intermediates. RESULTS: HEV particles consistent with LE, NLE, and an "intermediate" (IM) phenotype, obtained after S/D treatment, were seen in different HEV stocks. In the absence of HEV antibodies, log reduction factors (LRFs) of 4.0 and 2.5 were obtained by 35-nm nanofiltration of LE and IM HEV, consistent with the larger and smaller sizes of these phenotypes. Addition of HEV antibodies enhanced IM HEV removal around 1000-fold (LRF, 5.6). Effective (LRF, >4.8 and >4.0) HEV removal was obtained for the nanofiltration processing step for IG intermediates with varying HEV antibody content. CONCLUSION: HEV spikes used in clearance studies should be carefully selected, as differences in physicochemical properties might affect HEV clearance. Antibody-mediated enhancement of HEV nanofiltration was demonstrated in IG process intermediates even at low HEV antibody concentration, illustrating the robustness of this manufacturing step.

SARS-CoV-2 and the safety margins of cell-based biological medicinal products.

With the pandemic emergence of SARS-CoV-2, the exposure of cell substrates used for manufacturing of medicines has become a possibility. Cell lines used in biomanufacturing were thus evaluated for their SARS-CoV-2 susceptibility, and the detection of SARS-CoV-2 in culture supernatants by routine adventitious virus testing of fermenter harvest tested.

Immunoglobulins and virus antibody titers: of past needs, current requirements, and future options.

BACKGROUND: Immunoglobulins (Igs) have been in clinical use for almost 70 years, and early on were also used in conjunction with exposure to the measles virus or polio virus. The US regulations that describe functional Ig lot release thus require the demonstration of minimum antibody titers against these two viruses, although the use of vaccines has now dramatically reduced their incidence. The lower clinical importance of these viruses raises the question of whether other virus antibodies might be more informative for patients with immunodeficiency. STUDY DESIGN AND METHODS: A literature survey was conducted to identify viruses of potential clinical concern for people with immunodeficiency. The viruses selected have stable seroepidemiology and associated functional antibody assays. As a result, neutralizing antibody titers to human adenovirus 5 (HAdV5), respiratory syncytial virus (RSV) serotypes A and B, and human parainfluenza virus 3 (hPIV3) were determined in Ig lots produced from plasma collected in either the United States or the European Union. RESULTS: The virus antibody titers measured were high and consistent among the Ig lots tested. Use of either US- or EU-derived plasma as starting material resulted in equivalent virus antibody titers, with the exception of RSV serotype B, for which a lower titer was seen in EU plasma-derived Ig lots. CONCLUSION: With the significant decline in measles virus and polio virus circulation, and even their potential eradication, measurement of antibody titers against other viruses in Ig products may be more informative for functional lot release testing.

Continued use of poliovirus after eradication: hyper-attenuated strains as a safe alternative for release testing of human immunoglobulins.

BACKGROUND: Wild-type poliovirus may be eradicated soon and under WHO GAPIII guidance, laboratory use will be discontinued or subject to strict containment. Per US Code of Federal Regulations, however, immunoglobulin lot release testing will still require use of replicating poliovirus. The suitability of S19 hyper-attenuated and apathogenic poliovirus strains as alternatives to the currently used wild-type virus in such a release assay was investigated. STUDY DESIGN AND METHODS: S19 poliovirus strains were propagated in a commercial setting using good virological practices and maintenance of the S19 hyper-attenuated genotype was confirmed by massively parallel sequencing. RESULTS: The attenuated phenotype of the produced S19 stocks was confirmed in a highly sensitive mouse-model. Equivalency in performance was seen in the lot release assay for the S19 and wild-type polioviruses. CONCLUSION: The deployment of such hyper-attenuated and thoroughly characterized S19 stocks in these and other essential activities might reconcile final containment measures with continued safe use of poliovirus.

Measles Virus Neutralizing Antibodies in Intravenous Immunoglobulins: Is an Increase by Revaccination of Plasma Donors Possible?

We report a screen of plasma donors confirming that widespread use of childhood measles vaccination since 1963 resulted in a decrease in average measles virus antibody titers among plasma donors, which is reflected in intravenous immunoglobulins (IVIGs). The measles virus antibody titer, however, is a potency requirement for IVIGs, as defined in a Food and Drug Administration regulation. To mitigate the decline in measles virus antibody titers in IVIGs and to ensure consistent product release, revaccination of plasma donors was investigated as a means to boost titers. However, revaccination-induced titer increases were only about 2-fold and short-lived.

Zika virus is not thermostable: very effective virus inactivation during heat treatment (pasteurization) of human serum albumin.

BACKGROUND: The ongoing Zika virus (ZIKV) outbreak in the Americas has also raised concerns around the potential for ZIKV transmission via blood products. Plasma-derived products are considered safe, because effective viral-inactivation and removal methods are implemented in their manufacturing processes. However, a recent study has indicated that ZIKV is "thermally stable" compared with the closely related Dengue virus, thus raising the question of whether heat treatments, as embedded in the manufacturing of plasma-derived products, are as effective against ZIKV as was previously shown for other Flaviviruses. Therefore, the sensitivity of ZIKV to heat inactivation was investigated using the pasteurization of human serum albumin (HSA) as an example. STUDY DESIGN AND METHODS: Heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) of HSA was investigated for the capacity to reduce ZIKV in two different protein concentrations (5% and 25% HSA). The results were compared with data obtained in identical set-ups for the closely related West Nile virus, tick-borne encephalitis virus, and bovine viral diarrhea virus. RESULTS: Heat treatment of HSA inactivated ZIKV to below the limit of detection already during the heating phase to 57.0°C, that is, even before the 10-hour incubation at 58.0 ± 1.0°C commenced. For West Nile virus, bovine viral diarrhea virus, and tick-borne encephalitis virus, incubations up to 180 minutes were required to achieve inactivation to below the limit of detection. CONCLUSION: ZIKV was more sensitive to heat treatment than other members of the Flaviviridae and thus does not pose a concern for plasma products that include a heat treatment in their manufacturing process.

A nonenveloped virus with a lipid envelope: hepatitis A virus as used in virus-reduction studies.

BACKGROUND: Recently, a quasi-lipid-enveloped (LE) form of the traditionally nonlipid-enveloped (NLE) hepatitis A virus (HAV) was described in human serum and cell culture-derived HAV stocks. This discovery challenges the understanding of HAV reduction in virus clearance studies of plasma products, which were performed under the premise of an NLE nature of this virus. Here, the presence of LE particles in HAV stocks used for reduction studies was verified, and the hypothesis that LE and NLE particles might contribute to the differential heat sensitivity of HAV variants during heat treatment of human serum albumin was evaluated. STUDY DESIGN AND METHODS: Cell culture lysates and supernatants of two cytopathic HAV variants, HM175/18f and HM175/24a, were characterized for their LE and NLE particle content by isopycnic gradient centrifugation. The obtained fractions were characterized for relative infectivity and then subjected to heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) in 12.5% human serum albumin to investigate their respective heat sensitivity. RESULTS: Preparations of the two HAV variants contained either LE particles (HM175/24a) or LE and NLE particles (HM175/18f) with equivalent specific infectivity. For HM175/18f, heat sensitivity of LE and NLE fractions did not differ significantly, and inactivation of the whole virus stock was identical to the NLE particle inactivation profile, whereas the HM175/24a variant was more heat sensitive. CONCLUSION: The results indicate that, in heat-treatment studies, the LE or NLE HAV phenotype is less important than the choice of HAV variant, and the most heat-resistant HM175/18f should be used.

Hepatitis E virus and the safety of plasma products: investigations into the reduction capacity of manufacturing processes.

BACKGROUND: Hepatitis E virus (HEV) has been transmitted by transfusion of labile blood products and the occasional detection of HEV RNA in plasma pools indicates that HEV viremic donations might enter the manufacturing process of plasma products. To verify the safety margins of plasma products with respect to HEV, virus reduction steps commonly used in their manufacturing processes were investigated for their effectiveness to reduce HEV. STUDY DESIGN AND METHODS: Detection methods for HEV removal (by reverse transcription quantitative polymerase chain reaction) and inactivation (using an infectivity assay) were established. Immunoaffinity chromatography and 20-nm virus filtration for Factor (F)VIII, cold ethanol fractionation, and low-pH treatment for immunoglobulin, heat treatment for human albumin, and 35-nm nanofiltration for FVIII inhibitor-bypassing activity (FEIBA) were investigated for their capacity to reduce HEV or the physicochemically similar viruses feline calicivirus (FCV) and hepatitis A virus (HAV). RESULTS: For FVIII, HEV reduction of 3.9 and more than 3.9 log was demonstrated for immunoaffinity chromatography and 20-nm nanofiltration, respectively, and the cold ethanol fractionation for immunoglobulin removed more than 3.5 log of HEV, to below the limit of detection (LOD). Heat treatment of human albumin inactivated more than 3.1 log of HEV to below the LOD and 35-nm nanofiltration removed 4.0 log of HEV from the FEIBA intermediate. The results indicated HAV rather than FCV as the more relevant model virus for HEV. CONCLUSION: Substantial HEV reduction during processes commonly used in the manufacturing of plasma products was demonstrated, similar to that previously demonstrated for HAV.

Hyperimmune intravenous immunoglobulin containing high titers of pandemic H1N1 hemagglutinin and neuraminidase antibodies provides dose-dependent protection against lethal virus challenge in SCID mice.

BACKGROUND: Convalescent plasma and fractionated immunoglobulins have been suggested as prophylactic or therapeutic interventions during an influenza pandemic. FINDINGS: Intravenous immunoglobulin (IVIG) preparations manufactured from human plasma collected before the 2009 H1N1 influenza pandemic, and post-pandemic hyperimmune (H)-IVIG preparations were characterized with respect to hemagglutination inhibition (HI), microneutralization (MN) and neuraminidase-inhibiting (NAi) antibody titers against pandemic H1N1 (pH1N1) and seasonal H1N1 (sH1N1) viruses. The protective efficacy of the IVIG and H-IVIG preparations was evaluated in a SCID mouse challenge model.Substantial levels of HI, MN and NAi antibodies against pH1N1 (GMTs 1:45, 1:204 and 1: 727, respectively) and sH1N1 (GMTs 1:688, 1:4,946 and 1:312, respectively) were present in pre-pandemic IVIG preparations. In post-pandemic H-IVIG preparations, HI, MN and NAi antibody GMTs against pH1N1 were 1:1,280, 1:11,404 and 1:2,488 (28-, 56- and 3.4-fold enriched), respectively, compared to pre-pandemic IVIG preparations (p < 0.001). Post-pandemic H-IVIG (HI titer 1:1,280) provided complete protection from lethality of SCID mice against pH1N1 challenge (100% of mice survived for 29 days post-challenge). Pre-pandemic IVIG (HI titer 1:70) did not provide significant protection against pH1N1 challenge (50% of mice survived 29 days post-challenge compared to 40% survival in the buffer control group). There was a highly significant correlation between circulating in vivo HI and MN antibody titers and survival (p < 0001). CONCLUSION: The substantial enrichment of HA- and NA-specific antibodies in H-IVIG and the efficacious protection of SCID mice against challenge with pH1N1 suggests H-IVIG as a promising intervention against pandemic influenza for immunocompromised patients and other risk groups.