Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

The effect of dose-interval on antibody response to mRNA COVID-19 vaccines: a prospective cohort study.

Background: Vaccination against COVID-19 is highly effective in preventing severe disease and hospitalization, but primary COVID mRNA vaccination schedules often differed from those recommended by the manufacturers due to supply chain issues. We investigated the impact of delaying the second dose on antibody responses to COVID mRNA-vaccines in a prospective cohort of health-care workers in Quebec. Methods: We recruited participants from the McGill University Health Centre who provided serum or participant-collected dried blood samples (DBS) at 28-days, 3 months, and 6 months post-second dose and at 28-days after a third dose. IgG antibodies to SARS-CoV2 spike (S), the receptor-binding domain (RBD), nucleocapsid (N) and neutralizing antibodies to the ancestral strain were assessed by enzyme-linked immunosorbent assay (ELISA). We examined associations between long (≤89 days) versus short (<89 days) between-dose intervals and antibody response through multivariable mixed-effects models adjusted for age, sex, prior covid infection status, time since vaccine dose, and assay batch. Findings: The cohort included 328 participants who received up to three vaccine doses (>80% Pfizer-BioNTech). Weighted averages of the serum (n=744) and DBS (n=216) cohort results from the multivariable models showed that IgG anti-S was 31% higher (95% CI: 12% to 53%) and IgG anti-RBD was 37% higher (95% CI: 14% to 65%) in the long vs. short interval participants, across all time points. Interpretation: Our study indicates that extending the covid primary series between-dose interval beyond 89 days (approximately 3 months) provides stronger antibody responses than intervals less than 89 days. Our demonstration of a more robust antibody response with a longer between dose interval is reassuring as logistical and supply challenges are navigated in low-resource settings.

Construction of a Hantaan Virus Phage Antibody Library and Screening for Potential Neutralizing Activity.

China is one of the main epidemic areas for hemorrhagic fever with renal syndrome (HFRS). Currently, there is no human antibody specific to Hantaan virus (HTNV) for the emergency prevention and treatment of HFRS. To prepare human antibodies with neutralizing activity, we established an anti-HTNV phage antibody library using phage display technology by transforming peripheral blood mononuclear cells (PBMCs) of patients with HFRS into B lymphoblastoid cell lines (BLCLs) and extracting cDNA from BLCLs that secreted neutralizing antibodies. Based on the phage antibody library, we screened HTNV-specific Fab antibodies with neutralizing activities. Our study provides a potential way forward for the emergency prevention of HTNV and specific treatment of HFRS.

Neutralizing Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.

Decreased and Heterogeneous Neutralizing Antibody Responses Against RBD of SARS-CoV-2 Variants After mRNA Vaccination.

The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerging variants raises concerns about their capacity to evade immune protection provided by natural infection or vaccination. The receptor-binding domain (RBD) of the viral spike protein is the major target of neutralizing antibodies, and viral variants accumulate mutations in this region. In this study, we determined the antibody neutralization capacity against the RBD of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Epsilon (B.1.427), Kappa (B.1.617.1), and Delta (B.1.617.2) in a cohort of healthcare workers naturally infected or receiving COVID-19 mRNA vaccines from Moderna or Pfizer-BioNTech. We show that the five RBD variants displayed an augmented binding to ACE2 compared to the original Wuhan strain. The most significant increase was observed in variants Epsilon and Delta, containing mutation L452R. Using a flow cytometry cell-based assay, we found that SARS-CoV-2-infected subjects presented low levels of RBD-specific neutralizing antibodies against all variants analyzed, except Alpha. However, the neutralizing activity incremented considerably after a subsequent mRNA-vaccine dose, to levels significantly higher than those in naïve individuals receiving two vaccine doses. Importantly, we observed partially impaired neutralizing responses against most variants in fully vaccinated individuals. Variants Gamma and Kappa encompassing RBD E484K/Q mutations presented the highest neutralizing resistance. Furthermore, a wide heterogeneity in the magnitude of RBD-specific neutralizing responses against all tested SARS-CoV-2 variants following both mRNA vaccines was detected. Altogether, our findings provide important knowledge regarding SARS-CoV-2 vaccine-induced immunity, and should be very useful to guide future vaccination regimens and personalized vaccine approaches.

Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

Seroprevalence of Neutralizing Antibodies against Six Human Adenovirus Types Indicates the Low Level of Herd Immunity in Young Children from Guangzhou, China.

Human adenoviruses (HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3, -7, -4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population. Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies (NAb) against HAdV-3, -4, -7, -14, -55, and -11 in total 278 healthy populations between 0 months and 49 years of age (228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at 12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1-2, 3-5, and 6-17 years, respectively. The seroprevalence was very low (0% ~ 8.1%) for all other four types. In adults aged between 18 and 49 years, HAdV-3, -4, and -7 (> 50.00%) were the most common types, followed by HAdV-14 (38.00%), -55 (34.00%), and -11 (24.00%). Adults tended to have high NAb titers against HAdV-4 and -55. HAdV-55-seropositive donors tended to be HAdV-11- and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14, -55, -11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.

Disease Progression of Idiopathic Cervical Dystonia in Spite of Improvement After Botulinum Toxin Therapy.

Aim of the Study: To demonstrate general progression of symptoms in cervical dystonia (CD) on the one hand and improvement of some special symptoms on the other hand after botulinum toxin (BoNT) therapy. Methods: 74 patients with idiopathic CD under continuous treatment in a BoNT outpatient department with at least three injections, completed a short questionnaire. They were asked whether pain, increased muscle tone and tension, reduced mobility of the head, abnormal head position, head tremor, or other symptoms had been present at the onset of BoNT-therapy and which symptoms were present at the time of recruitment. Patients had to rate actual severity of CD in percent of the severity of CD at the onset of BoNT-therapy. The TSUI score was determined by the treating physician. Blood samples were taken to analyze induction of neutralizing antibodies. Results: Mean improvement of CD reported by the patients and scored by the physician was about 50%. The frequency of all symptoms increased with duration of therapy. The symptom most frequently improved was abnormal head position. The longer the time span between onset of symptoms and onset of BoNT-therapy was, the higher was the actual TSUI score and the lower the improvement reported. Twelve patients had positive antibody tests. Conclusions: Patients experience a progression of CD, but recognize improvement of abnormal head position due to BoNT-therapy. The longer patients have been without BoNT- therapy, the poorer is the long-term outcome independent on duration of BoNT treatment. Therefore BoNT-therapy should be initiated as early as possible.

Report on the AAPS Immunogenicity Guidance Forum.

In September 2018, the American Association of Pharmaceutical Scientists (AAPS) conducted an Annual Guidance Forum on the considerations related to immunogenicity testing for therapeutic protein products. In addition to a broad representation by the pharmaceutical industry, the event included strong representation by leading scientists from the US Food and Drug Administration (FDA). The agency and industry perspectives and updates to the guidance were presented. Specific topics that were discussed included the strategies of anti-drug antibody (ADA) assay cut-point assessments, the selection of ADA-positive controls (PCs), and the evaluation of PC performance. Assessment strategies and relevance of ADA assay attributes were also discussed, including assay drug tolerance and ADA assay sensitivity. The following is a summary of the discussion.