Assessment of the Biological Impact of SARS-CoV-2 Genetic Variation Using an Authentic Virus Neutralisation Assay with Convalescent Plasma, Vaccinee Sera, and Standard Reagents.

In the summer of 2020, it became clear that the genetic composition of SARS-CoV-2 was changing rapidly. This was highlighted by the rapid emergence of the D614G mutation at that time. In the autumn of 2020, the project entitled "Agility" was initiated with funding from the Coalition for Epidemic Preparedness Innovations (CEPI) to assess new variants of SARS-CoV-2. The project was designed to reach out and intercept swabs containing live variant viruses in order to generate highly characterised master and working stocks, and to assess the biological consequences of the rapid genetic changes using both in vitro and in vivo approaches. Since November 2020, a total of 21 variants have been acquired and tested against either a panel of convalescent sera from early in the pandemic, and/or a panel of plasma from triple-vaccinated participants. A pattern of continuous evolution of SARS-CoV-2 has been revealed. Sequential characterisation of the most globally significant variants available to us, generated in real-time, indicated that the most recent Omicron variants appear to have evolved in a manner that avoids immunological recognition by convalescent plasma from the era of the ancestral virus when analysed in an authentic virus neutralisation assay.

Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays.

Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.

Assessment of the protective effect of Imvamune and Acam2000 vaccines against aerosolized monkeypox virus in cynomolgus macaques.

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.