Background: Rabies remains a fatal zoonotic disease, necessitating effective and affordable vaccines. While current vaccines are effective, they require multiple doses and may not induce long-lasting immunity in all settings. The rabies virus glycoprotein (RABV-G) is the principal antigen responsible for eliciting virus-neutralizing antibodies, but its recombinant monomeric forms often suffer from poor immunogenicity due to misfolding and aggregation. Methods: A recombinant trimeric RABV-G ectodomain (rRABV-G-XVIII) was engineered by fusing it to a human collagen XVIII-derived trimerization domain. The protein was expressed in E. coli, purified under denaturing conditions, and refolded. Trimer formation was verified using size-exclusion chromatography. Mice were immunized with rRABV-G-XVIII, with or without adjuvant, and compared to a monomeric form (rRABV-GE). Antigen-specific antibody responses were measured by ELISA, neutralizing activity was assessed, and protective efficacy was evaluated via intracerebral challenge with the CVS-27 rabies strain. Results: rRABV-G-XVIII formed stable trimers and induced strong humoral immune responses, with high ELISA titers and virus-neutralizing activity comparable to an inactivated rabies vaccine. Mice immunized with rRABV-GE showed lower antibody responses and partial protection, which improved with adjuvant. All rRABV-G-XVIII-immunized mice were fully protected against rabies challenge, independent of adjuvant use. Conclusions: Stabilization of RABV-G in its native trimeric conformation markedly improves immunogenicity and protective efficacy. This approach offers a promising strategy for the development of rabies subunit vac-cines with simplified formulations and potential for cost-effective production in bacterial systems.

Latrodectus tredecimguttatus envenomation causes latrodectism, a neurotoxic syndrome driven primarily by α-latrotoxin (α-LTX)-mediated dysregulation of neurotransmitter release. This review examines the development and therapeutic application of specific immunoglobulins against L. tredecimguttatus toxins, integrating toxin diversity, α-LTX mechanisms, antibody production, preclinical neutralization, and clinical use. Eggs and spiderlings also contain bioactive proteins, including Latroeggtoxin-I and Latroeggtoxin-IV, although their relevance to human envenomation and antivenom development remains uncertain. α-LTX acts through receptor engagement, membrane pore formation, Ca²⁺ influx, and receptor-mediated signaling, providing potential targets for antibody neutralization. Conventional antivenoms rely mainly on animal-derived polyclonal immunoglobulins, whereas phage display and recombinant engineering enable fully human monoclonal antibodies with defined specificity and high affinity. Preclinical studies demonstrate neutralization across cellular, electrophysiological, ex vivo, and in vivo models, but methodological heterogeneity and variable cross-species activity complicate efficacy comparisons. Recombinant human antibodies offer improved molecular definition, reproducibility, and engineering potential, although clinical efficacy remains unestablished. Future development should prioritize standardized neutralization assays, clinically relevant post-exposure models, systematic cross-neutralization studies, and recombinant antibody combinations to achieve broader toxin coverage and safer, more standardized, reproducible, and clinically applicable immunotherapy for L. tredecimguttatus envenomation.

Efficient in vitro production of rabies virus is essential for vaccine development and quality control applications. High-density cultivation systems offer practical advantages for rabies virus production but also create culture conditions in which nutrient depletion, waste accumulation, and progressive deterioration of host-cell condition may limit infectious virus output. In this study, we evaluated the effects of sodium pyruvate supplementation on rabies virus CVS-11 production in Vero and BSR cells cultivated in a high-density macrocarrier-based tide-motion culture system under serum-containing and serum-free conditions, with complementary comparative observations in conventional monolayer cultures of BHK cells. Cultures were infected at a multiplicity of infection of 0.01, and infectious virus production was assessed over time, together with cell density, glucose consumption, and pH dynamics. Sodium pyruvate supplementation was associated with significantly higher infectious virus titers, delayed culture deterioration, prolonged maintenance of viable cell populations, and higher peak infectious titers in both Vero and BSR cultures. The highest infectious titers were observed under serum-free pyruvate-supplemented conditions, reaching 7.5 log₁₀ FFU/mL in Vero cells and 7.2 log₁₀ FFU/mL in BSR cells. Across the tested conditions, serum-free cultivation and pyruvate supplementation were both associated with significantly higher peak infectious titers. In contrast, exploratory correlation analysis based on condition-level summary values indicated an inverse association between minimum culture pH and peak infectious titer. Together, these findings show that sodium pyruvate supplementation can improve infectious rabies virus yield and prolong the productive phase in high-density macrocarrier-based cultures, supporting its use as a practical culture-modulation strategy for CVS-11 production in adherent cell systems.