The coronavirus disease 2019 (COVID‐19) pandemic has triggered a global health emergency and brought disaster to humans. Tremendous efforts have been made to control the pandemic, among which neutralizing antibodies (NAbs) are of specific interest to researchers. Neutralizing antibodies are generated within weeks after infection or immunization and can protect cells from virus intrusion and confer protective immunity to cells. Thus, production of NAbs is considered as a main goal for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccines and NAbs may be used for patient treatment in the form of monoclonal antibodies. Neutralization assays are capable of quantitatively detecting NAbs against SARS‐CoV‐2, allowing to explore the relationship between the level of NAbs and the severity of the disease, and may predict the possibility of re‐infection in COVID‐19 patients. They can also be used to test the effects of monoclonal antibodies, convalescent plasma and vaccines. At present, wild‐type virus neutralization assay remains the gold standard for measuring Nabs, while pseudovirus neutralization assays, Surrogate virus neutralization test (sVNT) and high‐throughput versions of neutralization assays are popular alternatives with their own advantages and disadvantages. In this review article, we summarize the characteristics and recent progress of SARS‐CoV‐2 neutralization assays. Special attention is given to the current limitations of various neutralization assays so as to promote new possible strategies with NAbs by which rapid SARS‐CoV‐2 serological diagnosis and antiviral screening in the future will be achieved.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in late 2019, laboratories around the world have widely used whole genome sequencing (WGS) to continuously monitor the changes in the viral genes and discovered multiple subtypes or branches evolved from SARS-CoV-2. Recently, several novel SARS-CoV-2 variants have been found to be more transmissible. They may affect the immune response caused by vaccines and natural infections and reduce the sensitivity to neutralizing antibodies. We analyze the distribution characteristics of prevalent SARS-CoV-2 variants and the frequency of mutant sites based on the data available from GISAID and PANGO by R 4.0.2 and ArcGIS 10.2. Our analysis suggests that B.1.1.7, B.1.351, and P.1 are more easily spreading than other variants, and the key mutations of S protein, including N501Y, E484K, and K417N/T, have high mutant frequencies, which may have become the main genotypes for the spread of SARS-CoV-2.

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Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have risen to dominance, which contains far more mutations in the spike protein in comparison to previously reported variants, compromising the efficacy of most existing vaccines or therapeutic monoclonal antibodies. Nanobody screened from high-throughput naïve libraries is a potential candidate for developing preventive and therapeutic antibodies. Methods Four nanobodies specific to the SARS-CoV-2 wild-type receptor-binding domain (RBD) were screened from a naïve phage display library. Their affinity and neutralizing activity were evaluated by surface plasmon resonance assays, surrogate virus neutralization tests, and pseudovirus neutralization assays. Preliminary identification of the binding epitopes of nanobodies by peptide-based ELISA and competition assay. Then four multivalent nanobodies were engineered by attaching the monovalent nanobodies to an antibody-binding nanoplatform constructed based on the lumazine synthase protein cage nanoparticles isolated from the Aquifex aeolicus (AaLS). Finally, the differences in potency between the monovalent and multivalent nanobodies were compared using the same methods. Results Three of the four specific nanobodies could maintain substantial inhibitory activity against the Omicron (B.1.1.529), of them, B-B2 had the best neutralizing activity against the Omicron (B.1.1.529) pseudovirus (IC 50 = 1.658 μg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC 50 = 0.653 μg/mL). The results of peptide-based ELISA indicated that LS-B-B2 might react with the linear epitopes in the SARS-CoV-2 RBD conserved regions, which would clarify the mechanisms for the maintenance of potent neutralization of Omicron (B.1.1.529) preliminary. Conclusion Our study indicated that the AaLS could be used as an antibody-binding nanoplatform to present nanobodies on its surface and improve the potency of nanobodies. The multivalent nanobody LS-B-B2 may serve as a potential agent for the neutralization of SARS-CoV-2 variants.

Recent exposure to seasonal coronaviruses (sCoVs) may stimulate cross-reactive antibody responses against severe acute respiratory syndrome CoV 2 (SARS-CoV-2). However, previous studies have produced divergent results regarding protective or damaging immunity induced by prior sCoV exposure. It remains unknown whether pre-existing humoral immunity plays a role in vaccine-induced neutralization and antibody responses. In this study, we collected 36 paired sera samples from 36 healthy volunteers before and after immunization with inactivated whole-virion SARS-CoV-2 vaccines for COVID-19, and analyzed the distribution and intensity of pre-existing antibody responses at the epitope level pre-vaccination as well as the relationship between pre-existing sCoV immunity and vaccine-induced neutralization. We observed large amounts of pre-existing cross-reactive antibodies in the conserved regions among sCoVs, especially the S2 subunit. Excep t for a few peptides, the IgG and IgM fluorescence intensities against S, M and N peptides did not differ significantly between pre-vaccination and post-vaccination sera of vaccinees who developed a neutralization inhibition rate (%inhibition) <40 and %inhibition ≥40 after two doses of the COVID-19 vaccine. Participants with strong and weak pre-existing cross-reactive antibodies (strong pre-CRA; weak pre-CRA) had similar %inhibition pre-vaccination (10.9% ± 2.9% vs. 12.0% ± 2.2%, P=0.990) and post-vaccination (43.8% ± 25.1% vs. 44.6% ± 21.5%, P=0.997). Overall, the strong pre-CRA group did not show a significantly greater increase in antibody responses to the S protein linear peptides post-vaccination compared with the weak pre-CRA group. Therefore, we found no evidence for a significant impact of pre-existing antibody responses on inactivated vaccine-induced neutralization and antibody responses. Our research provides an important basis for inactivated SARS-CoV-2 vaccine use in the context of high sCoV seroprevalence.

The diagnostic delays pose a huge challenge to human brucellosis (HB), which increases the risk of chronicity and complications with a heavy disease burden. This study aimed to quantify and identify the associated factors in the diagnostic delays to its prevention, reduction, and elimination. This study analyzed risk factors associated with the diagnostic delays in a cross-sectional study with data collected from Tongliao City, Inner Mongolia Autonomous Region of China. Diagnostic delays were defined with a cutoff of 30, 60, and 90 days. In different delay groups, risk factors of diagnostic delays were analyzed by univariate analysis and modeled by multivariate logistic regression analysis. A total of 14,506 cases were collected between January 1, 2005, and December 31, 2017, of which the median diagnostic delays was 29 days [interquartile range (IQR): 14–54 days]. Logistic regression analysis indicated that the older age category was associated with longer diagnostic delays across all groups. Longer diagnostic delays increase with age among three delay groups (p for trend <0.001). Occupation as herdsman was associated with shorter diagnostic delays in group 1 with 30 days [adjusted odds ratio (aOR), 0.890 (95% CI 0.804–0.986)]. Diagnostic delays was shorter in patients with brucellosis who were reported in CDC in all delay groups [aOR 0.738 (95% CI 0.690–0.790), 0.539 (95% CI 0.497–0.586), and 0.559 (95% CI 0.504–0.621)]. Pastoral/agricultural area was associated with shorter diagnostic delays in group 1 with 30 days [aOR, 0.889 (95%CI 0.831–0.951)] and group 3 with 90 days [aOR, 0.806 (95%CI 0.727–0.893)]. Stratified analysis showed that the older age category was associated with an increased risk of a long delay in both genders (p < 0.05). The older age group-to-youth group OR increased along with increased delay time (p for trend <0.001). Furthermore, the pastoral/agricultural area was associated with a shorter delay in males (p < 0.05). Delays exist in the diagnosis of HB. We should pay great attention to the risk factors of diagnostic delays, such as older population, non-herdsman, non-pastoral/agricultural area, non-disease prevention, and control agencies. Effective measures should shorten the diagnostic delays, achieve early detection, diagnosis, and treatment, and reduce the risk of HB's chronicity, complications, and economic burden.

The effect of cyclophosphamide on the pathogenesis of Marek's disease was examined in a line of chickens which is relatively resistant to Marek's disease. The injection of cyclophosphamide into newly hatched chickens delayed and reduced viremia and also reduced the development of Marek's disease lesions until 2 weeks after exposure to Marek's disease virus. The data indicate that a population of T cells susceptible to infection with virus and possibly viral transformation is affected by cyclophosphamide.