RADIOIMMUNOASSAY FOR DETECTION OF ANTI-RINDERPEST ANTIBODIES IN CATTLES

Liquid Phase Radioimmunoassay (RIA) was developed to detect and measure anti-rinderpest immunoglobulins in field animals sera, within two hours. Rinderpest virus adapted on Vero cell line culture and antigen purified by treatment with Triton-Gentron 13 Butanol, and, labeled with I isotope, using chloramin T iodination method. Comparative studies for detecting anti-rinderpest immunoglobulin in 80 calves sera samples, using the developed assay in parallel with virus neutralization test (VNT). The study showed 58.75 % agreement between the two methods. However, 71 % of seven months old, non-vaccinated calves showed anti rinderpest antibodies in their sera, also 81 % of 10 months old vaccinated calves were developed antibodies in their blood. These results demonstrate the development of sensitive, specific and rapid quantitative / qualitative radioimmunoassay, necessary for screening the development of immunity against rinderpest in cattle.

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a solid correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive ELISA formats or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) show excellent correlation to a cVNT with fully infectious SARS-CoV-2 and allow to estimate the reduced neutralization capacity of COVID-19 vaccinee sera with variants of concern of SARS-CoV-2.Author summaryThe current pandemic caused by SARS-CoV-2 is a major challenge not only for COVID-19 patients, medical staff, healthcare systems and the general public, but also virologists and clinical laboratories. A particular challenge are safety issues which require biological safety level 3 to work with and study the pathogen. An alternative are virus-like particles (VLPs) of SARS-CoV-2, which are authentic in terms of viral structure and function but are harmless bioproducts in nature. We engineered VLPs which are close-to-perfect mimics of SARS-CoV-2 by all structural, biochemical, physical and functional criteria tested. SARS-CoV-2 VLPs were used in virus neutralization tests (VNTs). Because high concentrations of neutralizing antibodies correlate with protection from COVID-19 practical VNTs are urgently needed. We developed an authentic, virus-free, thus safe yet very fast in vitro diagnostic test with SARS-CoV-2 VLPs. Virus neutralizing antibodies induced by natural infection or vaccination but also certain monoclonal antibodies inhibit VLP fusion with recipient cells carrying ACE2. Quantitative results from a conventional neutralization test with fully infectious SARS-CoV-2 and results from the VLP-based neutralization test correlate perfectly. The setup of the test is very flexible and allows to analyze sera for their neutralizing capacity against different variants of concern and in a standardized assay format.

Omicron infection enhances neutralizing immunity against the Delta variant

Omicron has been shown to be highly transmissible and have extensive evasion of neutralizing antibody immunity elicited by vaccination and previous SARS-CoV-2 infection. Omicron infections are rapidly expanding worldwide often in the face of high levels of Delta infections. Here we characterized developing immunity to Omicron and investigated whether neutralizing immunity elicited by Omicron also enhances neutralizing immunity of the Delta variant. We enrolled both previously vaccinated and unvaccinated individuals who were infected with SARS-CoV-2 in the Omicron infection wave in South Africa soon after symptom onset. We then measured their ability to neutralize both Omicron and Delta virus at enrollment versus a median of 14 days after enrollment. Neutralization of Omicron increased 14-fold over this time, showing a developing antibody response to the variant. Importantly, there was an enhancement of Delta virus neutralization, which increased 4.4-fold. The increase in Delta variant neutralization in individuals infected with Omicron may result in decreased ability of Delta to re-infect those individuals. Along with emerging data indicating that Omicron, at this time in the pandemic, is less pathogenic than Delta, such an outcome may have positive implications in terms of decreasing the Covid-19 burden of severe disease.

Homogeneous Surrogate Virus Neutralization Assay to Rapidly Assess Neutralization Activity of Anti-SARS-CoV-2 Antibodies

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a ‘Serological Assay based on a Tri-part split-NanoLuc® (SATiN)’ to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Herein, we expand on our previous work and describe a reconfigured version of the SATiN assay that can measure neutralization activity of antibodies directly from convalescent or vaccinated sera. The sensitivity is comparable to cell-based pseudovirus neutralization assays but with significantly shorter preparation and assay run time. As the assay is modular, we further demonstrate that Neutralization SATiN (Neu-SATiN) enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection

The emergence of the Omicron variant (1) of SARS-CoV-2 in November 2021 in South Africa has raised concerns that, based on the large number of mutations in the spike protein and elsewhere on the virus (https://covdb.stanford.edu/page/mutation-viewer/#sec_b-1-351), this variant will have considerable escape from vaccine elicited immunity. Furthermore, several mutations in the receptor binding domain and S2 are predicted to impact transmissibility and affinity for ACE-2. Here we investigated whether Omicron escapes antibody neutralization elicited by the Pfizer BNT162b2 mRNA vaccine and whether the virus still requires binding to the ACE2 receptor to infect cells. We used an early passage of isolated and sequence confirmed live Omicron virus isolated in South Africa. We used a human lung cell line clone (H1299-ACE2) engineered to express the ACE2 receptor (2) to both isolate the virus and test neutralization. We also tested growth in the parental H1299 which do not overexpress ACE2 and are not appreciably infectable with SARS-CoV-2 (Fig S1). The H1299-ACE2 cells were similar to Vero-E6 in titer dependent focus formation, but were considerably more sensitive (Fig S2). We observed that Omicron infected the ACE2-expressing cells in a concentration dependent manner but did not infect the parental H1299 cells, indicating that ACE2 is required for Omicron entry (Fig. 1A). We then tested the ability of plasma from BNT162b2 vaccinated study participants to neutralize Omicron versus ancestral D614G virus in a live virus neutralization assay. We tested 14 plasma samples from 12 participants (Table S1), with 6 having no previous record of SARS-CoV-2 infection nor detectable nucleocapsid antibodies indicative of previous infection. For two of these participants, we used samples from two timepoints. The remaining 6 participants had a record of previous infection in the first SARS-CoV-2 infection wave in South Africa where infection was with ancestral D614G virus (Table S1). Geometric mean titer (GMT) FRNT50 (inverse of the plasma dilution required for 50% reduction in infection foci number) was 1321 for D614G. These samples therefore had very strong neutralization of D614G virus, consistent with sampling soon after vaccination. GMT FRNT50 for the same samples was 32 for Omicron, a 41-fold decline (Fig 1B). However, the escape was incomplete, with 5 of the participants, all previously infected, showing relatively high neutralization titers with Omicron. Beta variant escape from BNT162b2 in a live virus neutralization assay has been reported to be substantial (3) and our own data confirmed these results (4), with about 3-fold reduction in FRNT50. The results we present here with Omicron show much more extensive escape. However, escape was incomplete in participants with higher FRNT50 due to previous infection. Previous infection, followed by vaccination or booster is likely to increase the neutralization level and likely confer protection from severe disease in Omicron infection.

Waning antibody levels after vaccination with mRNA BNT162b2 and inactivated CoronaVac COVID-19 vaccines in Hong Kong blood donors

Both inactivated vaccine (CoronaVac; Sinovac) and mRNA vaccine (Comirnaty/BNT162b2; Fosun-Pharma/BioNTech) are available in Hong Kong’s COVID-19 Vaccination Programme. We reported waning antibody levels by enzyme-linked immunosorbent assays (ELISA) and surrogate virus neutralization test (sVNT) among 850 fully vaccinated blood donors (i.e., received two doses). The BNT162b2 group’s antibody levels remain over the 50% protection threshold within six months, and the CoronaVac’s group’s median antibody levels begin to fall below the 50% protection threshold two months after vaccination.

Differences in Post-mRNA Vaccination SARS-CoV-2 IgG Concentrations and Surrogate Virus Neutralization Test Response by HIV Status and Type of Vaccine: a Matched Case-Control Observational Study

Following SARS-CoV-2 mRNA vaccination, people living with HIV (PLWH) had lower surrogate virus neutralization test response (p=0.03) and a trend towards lower IgG response (p=0.08), particularly among those with lower CD4+T-cell counts and who received the BNT162b2 vaccine. Study of the impact of supplemental vaccine doses among PLWH is needed.

Serum Neutralization Profiles of Straw-Colored Fruit Bats (Eidolon helvum) in Makurdi (Nigeria), against Four Lineages of Lagos Bat Lyssavirus

Lagos bat lyssavirus (LBV) comprising four lineages (A, B, C and D) can potentially cause the fatal disease rabies. Although LBV-B was initially isolated in Nigeria in 1956, there is no information on LBV lineages circulating in Nigeria. This study was undertaken for the first time to measure the neutralizing antibodies against four lineages of LBVs in straw-colored fruit bats (Eidolon helvum) in Makurdi, Nigeria. Serum samples (n = 180) collected during two periods (November 2017–March 2018 and November 2018–March 2019) from terminally bled bats captured for human consumption were tested using a modified fluorescent antibody virus neutralization (mFAVN) assay. A high proportion of bat sera (74%) neutralized at least one lineage of LBV (with reciprocal titers from 9 to >420.89) and most of them neutralized LBV-A (63%), followed by LBV-D (49%), LBV-C (45%) and LBV-B (24%). The majority of positive sera (75%, n = 100) neutralized multiple LBV lineages while the remaining 25% (n = 33) neutralized only a single lineage, i.e., LBV-A (n = 23), LBV-D (n = 8) and LBV-C (n = 2). None exclusively neutralized LBV-B. The results suggest that exposure to LBV is common in E. helvum and that LBV-A (but not LBV-B) is likely to be circulating in this region of Nigeria.