Human immune responses to the non-structural protein 3(NS3)and the envelope glycoprotein of Japanese encephalitis virus : interferon gamma and perforin production by NS3-specfic T cells correlate with protective immunity

Abstract

Immune Correlates of Protection and Diagnostic Antigen Development in Japanese Encephalitis Virus (JEV)

Abstract and Synopsis Introduction The flavivirus Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia, with ~35,000 cases and 10,000 deaths annually. Over half of survivors suffer lifelong neurological deficits such as paralysis, convulsions, memory loss, and speech/motor disturbances. Children under 10 years are the most affected. With no effective drug available, vaccination remains the only preventive strategy. Current vaccines, derived from infected mouse brains, are costly, risky to manufacture, and can cause allergic reactions, prompting efforts to develop recombinant vaccines.

Key Findings

Anticoagulant Studies

EDTA inhibited antigen-specific lymphoproliferative responses due to damage to antigen-presenting cells.

Heparin and EGTA preserved immune function, enabling reliable assays.

NS3-Specific T Cell Responses

NS3 protein was immunodominant in convalescent patients and healthy contacts.

Healthy individuals produced high levels of IFN- via both CD4 and CD8 T cells, correlating with protective immunity.

Patients showed deficient IFN- responses, correlating with severity of neurological sequelae.

NS3-specific CD8 T cells also produced perforin, indicating cytotoxic potential.

Immunodominant Epitope Mapping

Amino acids 193-324 of NS3 contained both class I and II epitopes.

This 14.4 kDa stretch elicited IFN- responses comparable to full-length NS3, making it an ideal vaccine candidate.

E Glycoprotein Analysis

E protein was not a dominant protective antigen.

IFN- responses to E were weak compared to NS3, highlighting NS3 as the stronger correlate of protection.

Diagnostic Antigen Development

Recombinant E glycoprotein ( E) was expressed in a baculovirus system without its membrane anchor.

Secreted E protein retained authentic antigenic properties, reacting with conformation-specific monoclonal antibodies.

Human sera confirmed E’s diagnostic potential, offering a safer alternative to mouse brain-derived antigens in MAC-ELISA.

Conclusions Protective immunity against JEV is strongly associated with NS3-specific Th1 responses and IFN- production.

The NS3 193-324 region is a promising candidate for T-cell-based vaccines.

The E glycoprotein is less relevant for protective immunity but remains useful for diagnostics.

Recombinant E protein expressed in baculovirus can replace conventional antigens in diagnostic assays, reducing cost and risk.

This thesis contributes significantly to both experimental vaccine development and diagnostic improvements for JEV, addressing urgent needs in endemic regions.