Investigating Cross-Reactive Immunity and Antibody-Dependent Enhancement (ADE) for Improved Vaccine Development

Investigating Cross-Reactive Immunity and Antibody-Dependent Enhancement (ADE) for Improved Vaccine Development Nur Hidayah Nor Isamuddin¹,3, Jalviana Lansayan¹,3, Kim Ling Chin2,3, Sazaly AbuBakar¹, Nurhafiza Zainal2* 1Tropical Infectious Diseases Research & Education Centre, Universiti Malaya, Kuala Lumpur, Malaysia 2Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia 3Institute for Advanced Studies, Universiti Malaya, Kuala Lumpur, Malaysia Cross-reactive immunity can influence susceptibility and immune responses to subsequent viral infections. While such immunity may confer protection through cross-neutralization, it can also trigger antibody-dependent enhancement (ADE), potentially worsening disease outcomes. Understanding these mechanisms is critical for designing safe and effective vaccines, particularly in regions where multiple viruses co-circulate. In this study, we examined the cross-reactive potential of anti-SARS-CoV-2 antibodies against influenza A virus (IAV) and two endemic arboviruses, chikungunya virus (CHIKV) and Japanese encephalitis virus (JEV). Archived serum samples from individuals with anti-SARS-CoV-2 IgG, anti-IAV IgG, anti-CHIKV IgG, or anti-JEV IgG, as well as samples with no detectable IgG to these viruses, were screened by ELISA. Cross‑neutralization was assessed using neutralization assays on MDCK cells for IAV and on Vero cells for CHIKV and JEV. ADE was evaluated using neutralization assays on Fc receptor‑bearing cells, with U937 cells for IAV and Raji B cells for CHIKV and JEV. Anti-SARS-CoV-2 antibodies lacked cross-neutralizing activity against IAV, CHIKV, and JEV but mediated ADE in IAV infection. In contrast, ADE in CHIKV and JEV infections was linked to prior exposure to their respective homologous arboviruses, not SARS-CoV-2. In IAV infection, ADE‑positive cultures showed increased MCP‑1 and RANTES and reduced IL‑10 and IFN‑β. In CHIKV and JEV infections, ADE‑exhibiting samples showed IL‑10 upregulation and TNF‑α downregulation. These findings underscore the importance of incorporating ADE risk assessment into vaccine development pipelines to ensure next-generation vaccines elicit protective immunity while minimizing enhancement risk.