Project Grant F31AI194693

ANTIGENIC DIVERGENCE AND FUNCTIONAL ANTIBODY RESPONSES IN EMERGING ORTHOPOXVIRUS CROSS-PROTECTION - ABSTRACT SINCE THE ERADICATION OF SMALLPOX IN 1980, MONKEYPOX (MPXV) HAS EMERGED AS THE MOST PROMINENT HUMAN ORTHOPOXVIRUS INFECTION GLOBALLY. HISTORICALLY CONFINED TO CENTRAL AND WEST AFRICA WITH PRIMARILY ZOONOTIC TRANSMISSION, RECENT EMERGING STRAINS HAVE DEMONSTRATED UNPRECEDENTED TRANSMISSION DYNAMICS CHARACTERIZED BY SUSTAINED HUMAN-TO-HUMAN TRANSMISSION AND GEOGRAPHIC SPREAD OUTSIDE AFRICA, EXEMPLIFIED BY CLADE IB MPXV'S RECENT LOCAL TRANSMISSION IN CALIFORNIA AS OF OCTOBER 2025. THE INCREASED LETHALITY OF CLADE IB COMPARED TO CLADE IIB FURTHER UNDERSCORES THE ESCALATING PUBLIC HEALTH THREAT POSED BY THESE EMERGING ORTHOPOXVIRUSES, ALONGSIDE OTHER DIVERGENT THREATS SUCH AS THE RECENTLY DISCOVERED BOREALPOX. DESPITE MAINTAINING A HIGH DEGREE OF GENETIC SIMILARITY ACROSS THE ORTHOPOXVIRUS GENUS, VACCINE MEDIATED CROSS-PROTECTION IS UNPREDICTABLE AND DECLINES WITH ANTIGENIC DISTANCE, LIMITING PANDEMIC PREPAREDNESS. MY PREVIOUS WORK DEMONSTRATES THAT CURRENT SMALLPOX VACCINES (MVA-BN) ELICIT ONLY MODERATE AND SHORT-LIVED ANTIBODY RESPONSES AGAINST ANTIGENICALLY DISTANT MPXV CLADE IIB, WITH NEUTRALIZATION CAPACITY DECLINING FURTHER AGAINST MORE DISTANT VIRUSES LIKE CLADE IB MPXV AND BOREALPOX. HOWEVER, THE MECHANISTIC BASIS OF CROSS-PROTECTIVE IMMUNITY WHEN NEUTRALIZING RESPONSES FAIL REMAINS UNDEFINED. TO DEFINE THE BREADTH AND LIMITATIONS OF ORTHOPOXVIRUS VACCINE PROTECTION, THIS PROPOSAL INCLUDES BOREALPOX AS THE MOST HIGHLY DIVERGENT HUMAN-INFECTING ORTHOPOXVIRUS, SERVING AS THE UPPER LIMIT FOR UNDERSTANDING CROSS-PROTECTIVE IMMUNITY ACROSS THE GENUS. THIS RESEARCH INVESTIGATES HOW ANTIGENIC DIVERGENCE ACROSS ORTHOPOXVIRUSES RELATES TO FUNCTIONAL ANTIBODY RESPONSES AND IDENTIFIES NON-NEUTRALIZING MECHANISMS THAT COMPENSATE WHEN NEUTRALIZATION IS REDUCED. AIM 1 CHARACTERIZES ANTIGENIC DIVERGENCE THROUGH SEQUENCE ANALYSIS AND IN SILICO EPITOPE PREDICTION, THEN VALIDATES FINDINGS USING RECOMBINANT PROTEIN ELISAS AND NEUTRALIZATION ASSAYS AGAINST VACV, CLADE IB MPXV, CLADE IIB MPXV, AND BOREALPOX. AIM 2 IDENTIFIES NON- NEUTRALIZING FC-EFFECTOR MECHANISMS (COMPLEMENT-MEDIATED NEUTRALIZATION AND FCGRIIIA-ADCC) THAT MEDIATE CROSS-PROTECTION WHEN BASELINE NEUTRALIZATION IS WEAK. INTEGRATION OF COMPUTATIONAL AND FUNCTIONAL DATA WILL DEFINE FUNCTIONAL IMMUNE PROFILES REVEALING HOW ANTIGENIC DISTANCE DRIVES SHIFTS FROM NEUTRALIZATION-DEPENDENT TO FC- EFFECTOR-DEPENDENT IMMUNITY. THESE FINDINGS WILL DIRECTLY INFORM RATIONAL VACCINE REDESIGN STRATEGIES FOR EMERGING ORTHOPOXVIRUS THREATS AND ENHANCE PANDEMIC PREPAREDNESS.