Monkeypox virus (MPXV) is in the genus Orthopoxvirus and is related to Variola virus (the virus that causes smallpox), and the causative agent of the multinational outbreak during 2022–2023. Although most mpox cases are self-limited and recover with supportive care, severe and fatal cases occur in persons with significant immunosuppression, most commonly advanced HIV infection. [1–5] Case descriptions include disseminated, coalescing, necrotizing lesions; involvement of multiple organ systems; and severe sepsis physiology. [1, 2] Disease can be protracted and paradoxical worsening after antiretroviral therapy (ART) initiation has raised concern for possible immune reconstitution inflammatory syndrome (IRIS), a dysregulated T-cell driven phenomenon. [2]

Management of severe, prolonged illness among patients with mpox and advanced HIV is challenging as it is unclear to what extent the disease is driven by virologic or inflammatory processes. Steroids, which are used to treat IRIS, are associated with severe illness and even death in animal models. [5, 6] Current recommendations for treatment of severe mpox emphasize early immune function optimization (e.g., ART for people with HIV) and early treatment with medical countermeasures active against orthopoxviruses (e.g., tecovirimat, brincidofovir, vaccinia immune globulin intravenous [VIGIV]). [5] To better understand disease pathogenesis in patients with advanced HIV, we evaluated tissues from skin biopsies and performed immunofluorescence and in situ hybridization (ISH) for MPXV mRNA to evaluate the distribution of virus in tissues and the local immune response.

In tissue biopsy samples from three patients with prolonged mpox illness and advanced HIV, high concentrations of MPXV were found in skin lesions and hair follicles. No MPXV-infected B cells were detected. These data suggest that prolonged mpox disease in people with advanced HIV is likely due to direct MPXV infection and ongoing replication, which may progress despite the use of currently available medical countermeasures.

Evidence suggests that B and T lymphocytes are critical to controlling MPXV and other orthopoxvirus infections. [11, 12] In orthopoxvirus infections, cytolytic CD4+ and CD8+ T-cells eradicate virus-infected monocytes and can minimize viral dissemination. [11] Animal model data indicate that CD4+ T-cell depletion before MPXV challenge reduces the development of protective B-cell and antibody responses and is associated with increased disease severity. [11, 13] HIV infection can mimic this effect through CD4+ T-cell deficiency with dysfunctional follicular helper T-cell support for B-cell maturation and antibody production and impairment of cytotoxic T-cells. [14]

In situ analysis of the skin lesions allowed us to establish localization, abundance, and type of immune cells present in the lesion material. Within the dermis and epidermis, infiltration of neutrophils (MPO+) in clusters and low numbers of CD4+ T-cells were observed. Within the upper dermis, right under infected epithelial cells, we detected a high density of myeloid and CD8+ cells (mainly dendritic cells and CD8+ T-cells). At sites of infection, recruitment, infiltration, and localization of these cell types may restrict lesion size by preventing viral spread. Additionally, within the mpox lesions, exclusively type 1 (MxA) interferon markers were expressed, suggesting a strong local innate immune response. [15] Despite the large number of CD8+ cells, neutrophil and myeloid infiltration, and an important type 1 IFN immune response, the level of MPXV-infected cells was high. Few B cells were present, and Langerhans cells were low or absent in lesion areas but were present within normal epidermis. Although we did not assess the functionality (killing and phagocytosing capabilities) of the immune cells present in lesions, findings indicate that the local immune response was insufficient to control MPXV replication.

Using RNAscope, we detected a remarkable number of cells harboring MPXV mRNA within the dermis and epidermis, as well as free virion within healthy area of the epithelium, suggesting that MPXV can be found outside of the lesions, as previously demonstrated during MPXV animal studies and in fatal mpox cases. [11, 16] In addition, two types of cells infected by MPXV were characterized: one identified as dendritic cells by DC-SIGN and CD1a expression, and the other identified as epithelial cells based on their localization and expression of the Pan-cytokeratin marker. MPXV-infected B cells were not detected. A high concentration of MPXV mRNA was found in the hair follicles, specifically in the external radicular sheath. These findings suggest the need for more aggressive antiviral approaches targeting viral replication and enhancing immune control mechanisms.

Despite extensive local MPXV infection and the presence of robust immune infiltrates, biopsy samples exhibited a scarcity of CD4+ T-cells, an observation consistent with depletion of circulating CD4+ T-cells due to advanced HIV. In immunocompetent patients with mpox, early expansion of activated effector CD4+ and CD8+ T-cells and a poxvirus specific Th1 cell response might protect against MPXV spread, persistence, and mutation. [15, 17] Additionally, intra-host MPXV genomic variability has been observed among patients with severe immunodeficiency that exhibited prolonged viral shedding. [17] In the United States, all 26 patients with confirmed tecovirimat resistance had HIV infection; among those with available data, all had low CD4+ T-cell counts. [18] Advanced HIV infection might favor viral persistence, compartmentalization, and the development of MPXV genomic mutations including those that may confer resistance to tecovirimat. [17, 18]

Limitations of our analysis include the small number of people and biopsy sites studied which may skew our findings, as well as the lack of additional controls including immune intact people with mpox who are unlikely to get skin biopsies for clinical purposes. Further evaluations are ongoing in the Virologic and Immunologic Characteristics of Severe Mpox Among Persons with Advanced HIV (VIRISMAP) study. [19]

In summary, we report large amounts of MPXV and type I interferon, with significant skin infiltration by innate immune cells such as dendritic cells, in patients with HIV and persistent mpox skin lesions. The lack of CD4+ T-cells and IFNγ in skin lesions indicates a lack of robust adaptive immune response in these patients. These findings highlight the need for antiviral treatment optimization in people with defective T-cell responses and suggest that IRIS might not be a significant component in persistent skin lesions of protracted mpox in persons with advanced HIV.