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.

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.

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.

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.

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.

Function matters: Coronavirus cross-binding antibodies do not cross-neutralize.

Background: During the current pandemic, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralization capacity of the immunoglobulin (IG) supply has changed from undetectable for lots manufactured from plasma collected before the pandemic, to now highly potent. Objective: As antibodies induced by exposure to or vaccination against coronaviruses were shown to be cross-coronavirus reactive, it was of interest to understand whether SARS-CoV-2 neutralizing antibodies would result in increased functional IG potency also against seasonal coronaviruses. Methods: IG lots from US plasma collected before SARS-CoV-2 emerged and collected during the pandemic were analyzed by live virus neutralization assay for SARS-CoV-2 and seasonal human coronaviruses (HCoVs) NL63 and OC43 neutralizing antibody content. Results: Pre-pandemic IG showed no SARS-CoV-2 neutralizing antibody titers. However, IG lots produced from plasma of post-coronavirus disease 2019 (COVID-19) individuals exhibited robust anti-SARS-CoV-2 potency (1,267 IU/ml) which further increased ~4-fold in pandemic IG lots reaching a mean titer of 5,122 IU/ml. Nonetheless, neutralizing antibody potencies to the HCoVs NL63 and OC43 remained stable over this period, i.e., have not increased correspondingly. Conclusion: The present results show that cross-coronavirus-reactive antibodies are not cross-neutralizing, i.e., SARS-CoV-2 antibodies do not neutralize seasonal coronaviruses NL63 and OC43.

SARS-CoV-2-Specific Antibody (Ab) Levels and the Kinetic of Ab Decline Determine Ab Persistence Over 1 Year.

In a SARS-CoV-2 seroprevalence study conducted with 1,655 working adults in spring of 2020, 12 of the subjects presented with positive neutralization test (NT) titers (>1:10). They were here followed up for 1 year to assess their Ab persistence. We report that 7/12 individuals (58%) had NT_50 titers ≥1:50 and S1-specific IgG ≥50 BAU/ml 1 year after mild COVID-19 infection. S1-specific IgG were retained until a year when these levels were at least >60 BAU/ml at 3 months post-infection. For both the initial fast and subsequent slow decline phase of Abs, we observed a significant correlation between NT_50 titers and S1-specific IgG and thus propose S1-IgG of 60 BAU/ml 3 months post-infection as a potential threshold to predict neutralizing Ab persistence for 1 year. NT_50 titers and S1-specific IgG also correlated with circulating S1-specific memory B-cells. SARS-CoV-2-specific Ab levels after primary mRNA vaccination in healthy controls were higher (Geometric Mean Concentration [GMC] 3158 BAU/ml [CI 2592 to 3848]) than after mild COVID-19 infection (GMC 82 BAU/ml [CI 48 to 139]), but showed a stronger fold-decline within 5-6 months (0.20-fold, to GMC 619 BAU/ml [CI 479 to 801] vs. 0.56-fold, to GMC 46 BAU/ml [CI 26 to 82]). Of particular interest, the decline of both infection- and vaccine-induced Abs correlated with body mass index. Our data contribute to describe decline and persistence of SARS-CoV-2-specific Abs after infection and vaccination, yet the relevance of the maintained Ab levels for protection against infection and/or disease depends on the so far undefined correlate of protection.

Feasibility of identifying plasma donors with high measles neutralizing antibody concentrations for the use of producing a measles hyperimmune globulin for postexposure prophylaxis.

Immune globulin (IG) is administered as measles postexposure prophylaxis (PEP) in people with primary immunodeficiency disorders or individuals not eligible for live virus vaccination. However, measles virus (MeV) neutralizing antibody (nAb) levels in plasma for fractionation and IG products fractionated thereof have declined. Here, the feasibility of producing a measles hyperimmune globulin (HIG) for PEP of high-risk individuals was investigated. Plasma samples (n = 384) were selected based on donor self-identification for previous MeV infection or vaccination, to determine the MeV-nAb content and compare it to the potency of plasma pools (n = 13) from the current IG manufacture. Convalescent donors have higher mean MeV-nAb concentrations (3.9 IU/mL) than vaccinated donors (2.5 IU/mL), as previously reported. However, their selection would only result in a 1.4-fold elevated nAb concentration compared to current plasma pools, which is not sufficient for HIG production. Interestingly, thirty-two donors (8%) had a MeV-nAb concentration of ≥ 8 IU/mL. The selective use of these plasma donations would result in sixfold higher plasma pool concentrations, which should permit the manufacture of the measles HIG. Further, the longitudinal analysis of a subset of individuals who repeatedly donated plasma at a high frequency revealed only a minor decline (~ 30%) of MeV-nAb levels. Repeat donations of such high-potency donors would thus facilitate the production of the measles HIG. Due to its markedly raised MeV-nAb concentration compared to standard IG, such preparation could significantly shorten infusion time and thus improve the treatment experience for both physicians and patients, especially infants.

Calibrated comparison of SARS-CoV-2 neutralizing antibody levels in response to protein-, mRNA-, and vector-based COVID-19 vaccines.

SARS-CoV-2 neutralizing antibodies have been suggested to reflect the efficacy of COVID-19 vaccines. This study reports the direct comparison of the SARS-CoV-2 neutralizing antibody response elicited by a protein- (NVX-CoV2373), an mRNA- (Comirnaty), and a vector-based (Vaxzevria) COVID-19 vaccine, calibrated against the WHO international SARS-CoV-2 antibody standard, and further supports the use of neutralizing antibody levels as a correlate of protection.

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.

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.

B and T cell response to SARS-CoV-2 vaccination in health care professionals with and without previous COVID-19.

BACKGROUND: In recent months numerous health care professional acquired COVID-19 at the workplace resulting in significant shortages in medical and nursing staff. We investigated how prior COVID-19 affects SARS-CoV-2 vaccination and how such knowledge could facilitate frugal vaccination strategies. METHODS: In a cohort of 41 healthcare professionals with (n=14) and without (n=27) previous SARS-CoV-2 infection, we assessed the immune status before, during and after vaccination with BNT162b2. The humoral immune response was assessed by receptor binding domain ELISA and different SARS-CoV-2 neutralisation assays using wildtype and pseudo-typed viruses. T cell immunity against SARS-CoV-2 surface and nucleocapsid peptides were studied using interferon-γ release assays and intracellular flow cytometry. Vaccine-related side effects were captured. FINDINGS: Prior COVID-19 resulted in improved vaccine responses both in the B and T cell compartment. In vaccine recipients with prior COVID-19, the first vaccine dose induced high antibody concentrations comparable to seronegative vaccine recipients after two injections. This translated into more efficient neutralisation of virus particles, even more pronounced than expected from the RBD ELISA results. Furthermore, T cell responses were stronger in convalescents and particularly strong against the SARS-CoV-2 nucleocapsid protein. INTERPRETATION: Herein, we corroborate recent findings suggesting that in convalescents a single vaccine dose is sufficient to boost adequate in vitro neutralisation of SARS-CoV-2 and therefore may be sufficient to induce adequate protection against severe COVID-19. New spike mutated virus variants render the highly conserved nucleocapsid protein - eliciting strong SARS-CoV-2 specific T cell immunity - an interesting additional vaccine target. FUNDING: Christian Doppler Research Association, Johannes Kepler University Linz.

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.

Neutralising SARS-CoV-2 RBD-specific antibodies persist for at least six months independently of symptoms in adults.

Background: In spring 2020, at the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Europe, we set up an assay system for large-scale testing of virus-specific and neutralising antibodies including their longevity. Methods: We analysed the sera of 1655 adult employees for SARS-CoV-2-specific antibodies using the S1 subunit of the spike protein of SARS-CoV-2. Sera containing S1-reactive antibodies were further evaluated for receptor-binding domain (RBD)- and nucleocapsid protein (NCP)-specific antibodies in relation to the neutralisation test (NT) results at three time points over six months. Results: We detect immunoglobulin G (IgG) and/or IgA antibodies reactive to the S1 protein in 10.15% (n = 168) of the participants. In total, 0.97% (n = 16) are positive for S1-IgG, 0.91% (n = 15) were S1-IgG- borderline and 8.28% (n = 137) exhibit only S1-IgA antibodies. Of the 168 S1-reactive sera, 8.33% (n = 14) have detectable RBD-specific antibodies and 6.55% (n = 11) NCP-specific antibodies. The latter correlates with NTs (kappa coefficient = 0.8660) but start to decline after 3 months. RBD-specific antibodies correlate most closely with the NT (kappa = 0.9448) and only these antibodies are stable for up to six months. All participants with virus-neutralising antibodies report symptoms, of which anosmia and/or dysgeusia correlate most closely with the detection of virus-neutralising antibodies. Conclusions: RBD-specific antibodies are most reliably detected post-infection, independent of the number/severity of symptoms, and correlate with neutralising antibodies at least for six months. They thus qualify best for large-scale seroepidemiological evaluation of both antibody reactivity and virus neutralisation.

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.

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.

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.