Establishing human 2D and 3D testicular models to elucidate monkeypox virus tropism and pathogenic mechanisms in the testes

The University of Hawaii at Manoa received a $156,500 Project Grant award from the National Institute of Allergy and Infectious Diseases (NIAID) under the Allergy and Infectious Diseases Research program (CFDA 93.855), effective August 1, 2025, through July 31, 2027. The project will establish human two-dimensional (2D) and three-dimensional (3D) testicular models to investigate monkeypox virus (MPXV) tropism and pathogenic mechanisms in the testes. This research addresses critical gaps in understanding MPXV's ability to infect the male reproductive tract and establish productive infections in genitourinary organs, particularly given confirmed reports of prolonged viral shedding in seminal fluid and emerging evidence of testicular involvement observed in nonhuman primate studies.

The funded research will leverage the University of Hawaii's existing 3D human testicular organoid (HTO) system—comprised of spermatogonia cells, Sertoli cells, Leydig cells, and peritubular myoid cells—to characterize direct evidence of MPXV infection in human testicular tissue, identify specific cellular targets of infection, and elucidate downstream pathogenic consequences. By combining established 2D culture systems with advanced 3D organoid models, the project will generate essential in vitro tools to examine viral-host interactions within the blood-testis barrier and assess potential reproductive complications, thereby supporting development of evidence-based transmission prevention strategies during the ongoing global MPXV outbreak.

Abstract

One of the most important lessons learned from the recent global outbreak of the monkeypox virus, now called mpox virus (mpxv, clade II), is the recognition that males are at a much higher risk for infection and higher occurrence of genital rash. Transmission via sexual contact is one of the main routes of virus spread. However, whether mpxv constitutes a sexually transmitted infection and can infect the male reproductive tract is still being debated, thus affecting the strategies to minimize transmission risk. Confirmed reports of mpxv shedding in seminal fluid for long after the clearance of viremia suggest the ability of mpxv to establish a productive infection in genitourinary organs. Poxviruses can also cause testicular complications, including azoospermia and seminiferous tubule atrophy. More recently, mpxv antigens have been detected in the testis of nonhuman primates both during the acute and convalescent stages, and the presence of testicular inflammation and necrosis in these macaques collectively suggests testis-tropism of mpxv, like zika and ebola viruses. However, direct evidence of mpxv infection in human testis is currently lacking, including cell targets of the virus and downstream consequences. Relevant human in vitro models are needed to characterize mpxv testicular infection. Human testis immune homeostasis is tightly governed by an elaborate communication network between different cells including testosterone-producing leydig cells (LC) and sertoli cells (SC) that form the blood-testis barrier (BTB). We recently established a 3D human testicular organoid (HTO) system comprised of undifferentiated spermatogonia cells, SC, LC, and peritubular myoid cells that closely recapitulates the cell diversity and function of the human testis to study zika virus and sars-COV-2 infection. We have also established 2D cultures of primary SC, LC, and mixed seminiferous tubule cells and an in vitro BTB model to delineate cell-specific responses to viruses. Therefore, the goal of this study is to utilize our 2D and 3D testicular culture systems as an effective in vitro surrogate to model testicular infection of mpxv and understand downstream consequences. In aim 1, we will assess mpxv infection in the 2D and 3D htos, identify cell targets, and characterize key infection pathologies, including cytopathic effects, antiviral response, and effect on BTB integrity. Aim 2 will utilize single-cell RNA sequencing to determine relative infectivity in each cell type and key pathways, including antiviral and inflammatory response, cell death, and spermatogenesis. Collective data will provide much-needed evidence of the testis as one of the target organs of mpxv replication after IT is cleared from blood and skin lesions and lay the foundation for future in vivo studies of transmission via the sexual route. The knowledge of whether mpxv is a sexually transmitted infection is critical in providing clinical management and transmission guidelines, especially in men who have SEX with men (MSM), an underrepresented group in biomedical research.