Characterization of an influenza virus pseudotyped with Ebolavirus glycoprotein

We have produced a new Ebola virus pseudotype: E-S-FLU, which can be handled in biosafety level-1/2 containment for laboratory analysis. E-S-FLU is a single cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza haemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU production. Infection of cells by E-S-FLU was dependent on Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. E-S-FLU was neutralized specifically by anti-Ebola glycoprotein antibody and a variety of small drug molecules that are known to inhibit entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC®1280 Sigma) of 1280 pharmacologically active compounds for inhibition of virus entry. 215 compounds inhibited E-S-FLU infection, while only 22 inhibited the control H5-S-FLU virus coated in an H5 haemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action e.g. calcium channel blockers, estrogen receptor antagonists, anti-histamines, serotonin uptake inhibitors etc. and this correlates with inhibitor screening results with other pseudotypes or wild-type Ebola virus in the literature. E-S-FLU is a new tool for Ebola virus cell entry studies and is easily applied to high throughput screening assays for small molecule inhibitors or antibodies.Importance Ebola virus is from the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates of Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus, and screen for inhibitors. Here we characterized a new surrogate named E-S-FLU, that is based on a disabled influenza virus core coated with the Ebola virus surface protein, but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU in biosafety level-1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small molecule library of 1280 candidates for inhibitors of viral entry

Use of the journal impact factor in academic review, promotion, and tenure evaluations

We analyzed how often and in what ways the Journal Impact Factor (JIF) is currently used in review, promotion, and tenure (RPT) documents of a representative sample of universities from the United States and Canada. 40% of research-intensive institutions and 18% of master’s institutions mentioned the JIF, or closely related terms. Of the institutions that mentioned the JIF, 87% supported its use in at least one of their RPT documents, 13% expressed caution about its use, and none heavily criticized it or prohibited its use. Furthermore, 63% of institutions that mentioned the JIF associated the metric with quality, 40% with impact, importance, or significance, and 20% with prestige, reputation, or status. We conclude that use of the JIF is encouraged in RPT evaluations, especially at research-intensive universities, and that there is work to be done to avoid the potential misuse of metrics like the JIF

Kinetics of immune responses to SARS-CoV-2 proteins in individuals with varying severity of infection and following a single dose of the AZD1222

To characterize the IgG and IgA responses to different SARS-CoV-2 proteins, we investigated the antibody responses to SARS-CoV-2 following natural infection and following a single dose of AZD1222 (Covishield), in Sri Lankan individuals. The IgG and IgA responses were assessed to S1, S2, RBD, and N proteins in patients at 4 weeks and 12 weeks since the onset of illness or following vaccination. Antibodies to the receptor-binding domain of SARS-CoV-2 wild type (WT), α, β, and λ and ACE2 (Angiotensin Converting Enzyme 2) receptor blocking antibodies were also assessed in these cohorts. For those with mild illness and in vaccines, the IgG responses to S1, S2, RBD, and N protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with moderate/severe illness. In the vaccines, IgG antibodies to the S2 subunit had the highest significant rise (P < 0.0001). Vaccines had several-fold lower IgA antibodies to all the SARS-CoV-2 proteins tested than those with natural infection. At 12 weeks, the haemagglutination test (HAT) titres were significantly lower to the α in vaccines and significantly lower in those with mild illness and in vaccines to β and for λ. No such difference was seen in those with moderate/severe illness. Vaccines had significantly less IgA to SARS-CoV-2, but comparable IgG responses those with natural infection. However, following a single dose vaccines had reduced antibody levels to the VOCs, which further declined with time, suggesting the need to reduce the gap between the two doses, in countries experiencing outbreaks due to VOCs

Seroprevalence of SARS-CoV-2 infection in the Colombo Municipality region, Sri Lanka

Background: As the Municipality Council area in Colombo (CMC) experienced the highest number of cases until the end of January 2021, in Sri Lanka, we carried out a serosurvey prior to initiation of the vaccination program to understand the extent of the SARS-CoV-2 outbreak. Methods: SARS-CoV-2 seropositivity was determined in 2,547 individuals between the ages of 10–86 years, by the Wantai total antibody ELISA. We also compared seroprevalence using the haemagglutination test (HAT) to evaluate its usefulness in carrying out serosurveys. Results: The overall seropositivity rate was 24.46%, while seropositivity by HAT was 18.90%. Although The SARS-CoV-2 infection detection rates by PCR were highest in the population between the ages of 20–60 years of age, there was no statistically significant difference in the seropositivity rates in different age groups. For instance, although the seropositivity rate was highest in the 10–20 age group (34.03%), the PCR positivity rate was 9.80%. Differences in the PCR positivity rates and seropositivity rates were also seen in 60–70-year-olds (8.90 vs. 30.4%) and in individuals >70 years (4.10 vs. 1.20%). The seropositivity rate of the females was 29.70% (290/976), which was significantly higher (p < 0.002) than in males 21.2% (333/1,571). Conclusions: A high seroprevalence rate (24.5%) was seen in all age groups in the CMC suggesting that a high level of transmission was seen during this time. The higher PCR positivity rates between the ages of 20–60 are likely to be due to increased testing carried out in the working population. Therefore, the PCR positivity rates, appear to underestimate the true extent of the outbreak and the age groups which were infected

Kinetics of immune responses to the AZD1222/Covishield vaccine with varying dose intervals in Sri Lankan individuals

Background To understand the kinetics of immune responses with different dosing gaps of the AZD1222 vaccine, we compared antibody and T cell responses in two cohorts with two different dosing gaps. Methods Antibodies to the SARS-CoV-2 virus were assessed in 297 individuals with a dosing gap of 12 weeks, sampled 12 weeks post second dose (cohort 1) and in 77 individuals with a median dosing gap of 21.4 weeks (cohort 2) sampled 6 weeks post second dose. ACE2-blocking antibodies (ACE2-blocking Abs), antibodies to the receptor-binding domain (RBD) of variants of concern (VOC), and ex vivo T cell responses were assessed in a subcohort. Results All individuals (100%) had SARS-CoV-2-specific total antibodies and 94.2% of cohort 1 and 97.1% of cohort 2 had ACE2-blocking Abs. There was no difference in antibody titers or positivity rates in different age groups in both cohorts. The ACE2-blocking Abs (p Conclusions Both dosing schedules resulted in high antibody and T cell responses post vaccination, although those with a longer dosing gap had a higher magnitude of responses, possibly as immune responses were measured 6 weeks post second dose compared to 12 weeks post second dose

Comparison of the immunogenicity of five COVID-19 vaccines in Sri Lanka

To determine the antibody responses elicited by different vaccines against SARS-CoV-2, we compared antibody responses in individuals 3 months post-vaccination in those who had received different vaccines in Sri Lanka. Abs to the receptor binding domain (RBD) of the ancestral (wild type) virus (WT) as well as to variants of concern (VoCs), and ACE2 blocking Abs, were assessed in individuals vaccinated with Moderna (n = 225), Sputnik V (n = 128) or Sputnik light (n = 184) and the results were compared with previously reported data on Sinopharm and AZD1222 vaccinees. A total of 99.5% of Moderna, >94% of AZD1222 or Sputnik V and >70% of Sputnik light, >60% of Sinopharm vaccine recipients, had a positive response to ACE2 blocking antibodies. The ACE2 blocking antibody levels were highest to lowest was Moderna > Sputnik V/AZD1222 (had equal levels) > Sputnik light > Sinopharm. All Moderna recipients had antibodies to the RBD of WT, alpha and beta, while positivity rates for delta variant was 80%. The positivity rates for Sputnik V vaccinees for the WT and VoCs were higher than for AZD1222 vaccinees while those who received Sinopharm had the lowest positivity rates (<16.7%). The total antibodies to the RBD were highest for the Sputnik V and AZD1222 vaccinees. The Moderna vaccine elicited the highest ACE2 blocking antibody levels followed by Sputnik V/AZD1222, while those who received Sinopharm had the lowest levels. These findings highlight the need for further studies to understand the effects on clinical outcomes

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80–99 years, n = 89) and younger adults (23–77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1–89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72–0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72–76%) older adults respond after two vaccinations to alpha and delta, but only 58–62% to beta and gamma, compared to 96–97% of younger vaccinees and 68–76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.publishedVersio

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world

Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants