Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals.

Rabies is an ancient fatal disease with no other available treatment than post-exposure vaccination, where the bite of infected animals, mainly dogs, is the leading cause of its transmission to human beings. In this context, global vaccination campaigns of companion animals, as well as wildlife reservoirs vaccination, are key factors to achieve the "Zero by 30" plan that pursues the eradication of dog-mediated human rabies by 2030. Rabies virus-neutralizing antibodies (VNAs) play an essential role in the disease protection, as it correlates with an adequate immune response and allows evaluating pre-or post-exposure prophylaxis efficacy. Hence, counting with reliable, accurate, and robust serological tests is of paramount importance. Currently, RFFIT and FAVN are the gold standard VNAs tests recommended by both the WHO and the OIE. Despite these methodologies are efficient and widely used, they present several drawbacks, as they are less easily to standardize and require the use of live rabies virus, containment facilities, and skilled professionals. Thus, in this review, we describe the state-of-the-art of alternative analytical methodologies currently available for rabies serology, with novel approaches based on pseudotyped recombinant viruses and emphasizing in the antigen binding methodologies that detect and quantify antibodies against the rabies glycoprotein. We discussed the wide range of assays that are interesting tools for a faster measurement of anti-rabies glycoprotein antibodies and, in some cases, less complex and more versatile than the gold standard methods. Finally, we discussed the key issues during the design and optimization steps of ELISA assays, highlighting the importance of validation and standardization procedures to improve rabies serology tests and, as a consequence, their results. Key points• An exhaustive revision of rabies serology testing was made.• No rabies serology assay can be thought as better than others for all intents and purposes.• The validation procedure guarantees reliable and consistent results among the globe.

HIGHLIGHTS Roller bottle sustain the continuous production of Rabies Virus-Like Particles (RV-VLP).  Roller bottle produced RV-VLPs induce a specific antibody response in mice.  RV-VLPs without adjuvant addition induce high titer of long-lasting neutralizing antibodies.  Potency test of the vaccine candidate demonstrate protection against rabies virus challenge.  RV-VLPs could be the basis for a simplified platform for a rabies veterinary vaccine.

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Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. Key points • Adequate regions for foreign epitope display in RVG were found. • G-H loop of FMDV was inserted in three regions of RVG. • The foreign epitope was detected by specific antibodies on fusion proteins. • G-H loop was detected on the surface of chimeric VLPs.