How are neutralizing antibodies different from binding antibodies?
In the case of any virus/bacterial infection, antibodies are generated by B-cells as a part of the humoral immune response of our system. But not all the generated antibodies which bind the pathogenic part of the foreign particle are able to inhibit the infectivity of the pathogen or vector, it is only a fraction of them which can interfere and block their entry and infectivity and those are neutralizing antibodies (NAb).
NAb detection is important
Precise assessment of neutralizing antibody activities is important for infected patients and for animals and volunteers immunized with the experimental vaccines against the antigens for the infecting virus/bacteria. Whether a vaccine can elicit a rigorous immune response in terms of NAb or not, decides the measure of its potency and protectivity. Also, the preexisting NAbs which represent the adaptive immune response of an individual previously exposed to antigen/virus can interfere with efficacy and risk assessment of a biotherapeutics. Therefore, it is critical to design reproducible and quantitative in vitro virus neutralization assays.
Development of NAb assay
Virus neutralization is a specialized type of immunoassay, where we measure the ability of antibody to block the infectivity of the virus. Reporter cells are engineered to express reporter genes for ex firefly luciferase under the control of the desired virus LTR permitting sensitive and accurate measurements of infection. Expression of the reporter genes is induced by viral proteins soon after infection. Luciferase activity is quantified by luminescence and is directly proportional to the number of infectious virus particles present in the test sample. If the test serum/ plasma has neutralizing antibodies, they will neutralize the virus and it will not turn on the luminescence in the cells. Antibody titer is obtained by identifying the serum dilution point at which blocking of virus activity is < 50% (Figure 1)
Considerations and challenges for NAb assay design
Neutralization assays could be designed either in vitro or in vivo.
- In vivo assays require embryonated eggs or animals. Although in vivo assays could be the ideal model for quantitative analyses of neutralization antibodies for evaluation of the potential vaccine or antiviral agents. But In vivo evaluation of antivirals involves monitoring the survival rate of the animals following lethal challenges with the wt viruses or with antivirals. This requires a long time and exhibits variability due to differences in immune response amongst animals and the consistency across the programs.
- The in vitro assays are cell-based assays. Cell-based assays are better and preferred as they provide a functional readout using relevant cell lines, which closely reflects the in vivo However, cell-based NAb assays are difficult to establish, as suitably relevant cell lines and a proper assay endpoint have to be identified and the sensitivity of the assay is difficult to establish. While completely protected cells can be easily distinguished from the damaged cells, partially protected cell populations are hard to evaluate. Therefore, it is difficult to come up with a titration curve to measure the strength of a neutralizing antibody with serially diluted testing antibodies.
- Types of viral vector: There can be either lentiviral or AAV transduction vectors. AAV serotypes are known to have tissue tropism hence an ideal cell line that allows AAV transduction is needed.
- NAb assay development for clinical assays/trials is challenging as it must be qualified and validated for human use.
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