In vitro assessment of foot-and-mouth disease vaccine immunogenicity: advances, challenges, and ethical considerations

Figures

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  Figure 1. Schematic representation of the genomic organization of the foot-and-mouth disease virus (FMDV). The positive-sense single-stranded RNA genome (~8.4 kb) encodes a single large open reading frame (ORF) of ~7,000 nucleotides flanked by structured untranslated regions (UTRs). The 5′ UTR contains an internal ribosome entry site (IRES) that enables cap-independent translation initiation, while the 3′ UTR contributes to genome replication and stability (90). The ORF encodes a single polyprotein, cleaved into four structural proteins (VP1–VP4) that assemble into a pseudo-T = 3 icosahedral capsid (~25–30 nm in diameter) and act as key antigenic targets for immune recognition. The polyprotein is also processed into 8–10 non-structural proteins (Lpro, 2A, 2B, 2C, 3A, 3B1-3, 3Cpro, and 3Dpol) that mediate viral replication and immune evasion.

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  Figure 2. Schematic of the virus neutralization test (VNT) assay workflow. This diagram illustrates the sequential process of detecting functional, virus-neutralizing antibodies in test samples. In this procedure, serum samples from previously vaccinated or FMDV-exposed animals are serially diluted or titrated down the multi-well culture plate to assess the level of neutralizing antibodies. A standardized reference virus antigen, typically derived from field isolates propagated in permissive cell lines such as BHK-21 (baby hamster kidney) or lamb kidney cells is added to each well at a defined infectivity dose, commonly 100 TCID50 per well. The degree to which the serum antibodies neutralize the virus reflects the immune response elicited by vaccination or infection. In the context of vaccine evaluation frameworks, VNT remains a gold standard, as it quantitatively measures the functional antibody response and helps determine whether a vaccine induces a sufficient level of protective immunity.

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  Figure 3. Stepwise illustration of the DIVA ELISA used to detect antibodies against FMDV non-structural proteins (NSP). The illustration depicts interactions in a multi-well culture plate pre-coated with FMDV NSP antigen, captured by a 3ABC-specific monoclonal antibody. Serum samples from test animals are added and may contain antibodies against the 3ABC antigen if the animal has been naturally infected with FMDV. These antibodies compete with a HRP-labeled detection monoclonal antibody for binding to the immobilized antigen. Following washing steps, the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) is added. The resulting color intensity measured by absorbance at 450 nm reflects the concentration of anti-NSP antibodies in the serum (91). Within vaccine evaluation and surveillance frameworks, NSP antibody ELISAs are crucial for distinguishing between vaccinated and infected animals, particularly in regions using purified, inactivated vaccines that do not elicit NSP responses. This differentiation is essential for accurately monitoring vaccine performance and verifying that serological responses result from vaccination rather than field virus exposure, thereby ensuring the…

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  Figure 4. Schematic representation of cell-based (cytotoxicity or cytopathogenicity) assays. This illustration depicts the process of detecting FMDV in tissue and serum samples using infection-susceptible lamb kidney primary cells cultured in growth media. The procedure entails cell culture preparation and proliferation in flasks to a confluent monolayer. Epithelial tissue, probang (oropharyngeal fluid) or serum samples which may contain FMD virus are inoculated onto the cell monolayer and incubated. The plates are examined under a microscope after 48 h to determine the presence of CPE. The presence of the virus would destroy the monolayer due to viral replication whereas the absence of the virus would be identified by an intact monolayer.

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  Figure 5. Schematic illustration of reverse transcription polymerase chain reaction (RT-PCR) workflows for FMD diagnosis. (A) Two-step RT-qPCR: Viral RNA is first reverse transcribed into complementary DNA (cDNA) using reverse transcriptase and appropriate primers, followed by amplification in a separate quantitative PCR (qPCR) reaction using sequence-specific primers and DNA polymerase. (B) One-step RT-qPCR: Reverse transcription and qPCR amplification are performed simultaneously in a single reaction tube containing reverse transcriptase, DNA polymerase, primers, and reaction buffer components. The amplification curves illustrate fluorescence-based detection during qPCR, with cycle threshold (Ct) values inversely related to viral RNA concentration.