Advances in Surrogate Neutralization Tests for High-Throughput Screening and the Point-of-Care
Creators: name: Streif, Simon
name: Baeumner, Antje J.
Publisher: name: Universität Regensburg
Year: 2025
Subjects: subject: 540 Chemie

description: Serological testing has long played a crucial role in disease management and clinical diagnostics. Both infection with viruses and vaccination mediate a humoral immune response, including the generation of specific antibodies. The presence of antibodies can therefore be used qualitatively to detect recent or past infections or quantitatively to determine the immune status of a patient. Antibody profiles generated by different viruses vary and show differences for infection versus vaccination, as the latter often uses only one specific antigen rather than the whole virus. Furthermore, while some vaccinations result in long-term immunity, others require booster shots every few years, or even yearly. Hence, the necessity and benefits of serological testing are typically tailored to the respective viruses or diseases.
Diseases preventable through vaccination include, e.g., hepatitis A, influenza, SARS, chickenpox, measles, mumps, rubella, tetanus, and poliomyelitis. Low mutation rates due to constraints, such as limited host range in the case of measles, (1) are key to ensuring long-term immunity by memory B cell and T cell persistence. Vaccination can lead to immunity for up to 30 years and longer for the hepatitis B virus. (2) The influenza viruses, especially influenza A, show rapid antigenic drift, necessitating extensive modeling to predict the most likely strains to be targeted by the annual vaccine. (3) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also shows high mutation rates, facilitating its immune escape. The COVID-19 pandemic caused by the SARS-CoV-2 virus sparked advancement and innovation in the field of serological testing with regard to both binding and neutralizing antibody detection. The virus consists of four proteins: nucleocapsid (N), envelope (E), membrane (M), and spike (S). Interaction with the host cell is mediated by the S protein as the receptor binding domain (RBD) of the S1 subunit binds to the human angiotensin converting enzyme 2 (ACE2) receptor. (4) Cellular transmembrane protease serine 2 (TMPRSS2) and lysosomal cathepsin proteases cleave the S1 and S2 subunits, followed by membrane fusion initiated by the S2 subunit. (5) While some antibodies target the nucleocapsid protein, the majority are directed against the spike protein, more specifically RBD. (6,7) Most SARS-CoV-2 vaccines make use of this observation by introducing mRNA or viral vectors to induce the expression of the S protein as the antigen (8,9) or by directly introducing the S protein. (10) Testing for antinucleocapsid antibodies can thus be used to check for past infections, unless an inactivated vaccine was used, introducing all viral proteins. (11) Antispike and anti-RBD antibodies, which are produced after both infection and vaccination, provide a means to assert the immune status and might serve as a correlate of protection (CoP). (12−14) For more information on the definition of a CoP for SARS-CoV-2 and other viruses, the reviews by Perry et al., (14) Sobhani et al., (15) and Plotkin (16) are recommended.
Two categories of antibodies can be quantified, binding antibodies and neutralizing antibodies (nAbs). The former includes all antibodies directed against a certain antigen, while the latter includes only antibodies that prevent infection, i.e., by blocking the virus–host interaction or preventing the host–cell fusion. Neutralization tests mimic the interaction of the virus with the host cell and thus quantify the neutralizing antibodies indirectly via their ability to block the interaction. The gold standard is the plaque reduction neutralization test (PRNT), which uses live virus incubated with patient serum dilutions prior to the addition to cells expressing the respective viral receptor. Infection of the cells by the virus results in the formation of plaques, which are quantified by manual or automatic counting. The PRNT50 value correlates to the serum dilution required to reduce the plaque formation observed without serum by 50%. The use of live virus makes the PRNT and other conventional neutralization tests (cVNTs), such as the micro neutralization test (microNT), highly accurate but requires a biosafety level (BSL) facility of the virus and results in long turn-around times of up to 3 days. (17) In the case of the viruses mentioned above, BSL-3 would be required for most. To reduce the safety requirements to at least BSL-2, pseudovirus-neutralization tests (pVNTs) have been developed, relying on the use of lentiviruses or vesicular stomatitis virus pseudotyped with the respective viral protein responsible for host cell binding and fusion. (18−20) For more information on live virus neutralization test and pVNT development, the recent reviews of Rocha et al., (21) Sun et al., (22) and Vaidya (23) are recommended. In the case of SARS-CoV-2, RBD was identified as the main target for neutralizing antibodies, providing the opportunity to further simplify the neutralization tests. Surrogate virus neutralization tests (sVNTs), the focus of this review, rely on the competitive binding of neutralizing antibodies and the cell receptor with the relevant viral protein. In the case of SARS-CoV-2, this is ACE2 and RBD, respectively. These cell-free assays can be divided into two categories: high-throughput screening (HTS) and point-of-care (POC) assays. They do not require any biosafety facilities and have rapid turn-around times, making serological testing widely available. The COVID pandemic showed that such tests can be applied to monitor the development of antibody titers in vaccination studies, providing insights into the immunity against SARS-CoV-2. Still, such sVNTs are not endorsed by regulatory agencies to monitor the immune status, yet. (24) To date, only three sVNTs have been granted emergency use authorization (EUA) by the Food and Drug Administration (FDA). (25) Lacking standardization and validation during test development as well as difficulties defining a neutralizing antibody titer as CoP due to the rapid mutation of the virus currently hinders progress to take full advantage of sVNTs. However, they are the scientific and technological answer to broad serological testing needed and not only in pandemic situations. In the following, an overview of the development in the field of sVNTs of the last three years is provided. Advantages and disadvantages of the different formats are critically analyzed, and future development potential, especially the applicability toward other viruses, is discussed.