LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox

Abstract

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.

FIG. 1.

Local cutaneous lesions at the site (upper left arm) of vaccination with Lister or LC16m8. (A) Typical vaccine-induced local lesions on the designated days postimmunization. Bars, 10 mm. (B) Sizes (areas) of the vaccine-induced lesions with Lister (n = 5) or LC16m8 (n = 6), measured on day 13 and shown as averages and standard deviations.

FIG. 2.

Changes in body weight, MPXV loads in total peripheral blood, and cytokine responses. (A) Body weight expressed as a percentage of that measured at the time of MPXV challenge. (B) Viral loads in total peripheral blood as measured by qPCR. (C) IFN-γ response. (D) IL-6 response. Left and right panels show these indicators for monkeys challenged intranasally with MPXV strain Liberia and subcutaneously with MPXV Zr-599, respectively.

FIG. 3.

Macroscopic and histological lesions observed in naïve monkeys infected with MPXV. (A) Macroscopic and histological lesions in the skin, lungs, and pancreas in the IN-Naïve group. Papulovesicular lesions were observed in the skin (a), and nodular and granulomatous lesions were present in the lungs (b) and pancreas (c). (d) The edges of the cutaneous lesions were characterized by epithelial cell swelling, epidermal hyperplasia, hyperkeratosis, necrosis, and infiltration of inflammatory cells. (e) Nodular and granulomatous lesions in the lungs were characterized by destruction of alveolar structures, necrosis, edema, proliferating fibroblasts, and infiltration of inflammatory cells. (f) Nodular and granulomatous lesions in the pancreas were characterized by extensive necrosis with infiltration of inflammatory cells and proliferating fibroblasts. (g to i) In these lesions, MPXV antigens were demonstrated by IHC analyses, indicating that they were caused by MPXV infection. (B) Macroscopic and histological lesions in the thymus, stomach, and colon in the SC-Naïve group. (a to c) Multiple nodular lesions were present in the thymus, and papilliform and granular lesions with hemorrhaging were seen in the lumens of the stomach and colon. (d) The lesions in the thymus were characterized by granulomatous inflammation and proliferation of fibrous tissue consisting of fibroblastic cells, histiocytes, and microvascular structures. (e) The histology in the stomach consisted of necrotic changes with inflammatory cells including neutrophils. (f) The submucosal area of the colon consisted of fibroblastic tissues with granulomatous inflammation characterized by infiltration of inflammatory cells. The mucosal membranes showed ulceration. (g to i) In these lesions, MPXV antigens were demonstrated by IHC analyses, indicating that they were caused by MPXV infection.

FIG. 4.

Histology of the nasal cavity caused by intranasal challenge with MPXV strain Liberia (A) and macroscopic lesions at the site of subcutaneous inoculation with MPXV strain Zr-599 (B). The identification numbers of monkeys are given. Analyses by H&E staining (A, low and high magnifications) revealed that the lesions of naïve monkey 4595 were characterized by destruction of mucous membrane structures, disappearance of mucosal epithelial cells resulting in ulcer formation, necrosis, and hyperplasia. MPXV antigens were present in the lesions. In contrast, the mucous membranes of the nasal cavity into which MPXV was inoculated in Lister-immunized monkey 4597 were normal, and MPXV antigens were not detected. Although the mucous membranes of the LC16m8-immunized monkey 4600 showed infiltration of inflammatory cells, the structure was maintained without necrosis. Furthermore, MPXV antigens were not detected. (B) Erythematous, vesicular, and ulcerative lesions appeared in the SC-Naïve group. The maximum diameter of the lesions exceeded 10 cm on day 14 postchallenge. In the SC-LC16m8 group, similar but milder lesions were observed, while no obvious lesions were detected at the site of inoculation in the SC-Lister group.

FIG. 5.

Vaccinia virus-specific IgG responses determined by IgG ELISA (A) and a neutralizing assay (B) for MPXV. The optical densities at 405 nm (OD₄₀₅) at serum sample dilutions of 1:100 are shown. (A) Development of specific IgG antibody responses to vaccinia virus antigens in plasma samples collected on different days after vaccination and/or challenge, as measured by IgG ELISA. The day of MPXV challenge was taken as day zero. (B) MPXV-specific neutralizing antibody titers (NT) in plasma samples were determined by plaque reduction assay on different days after vaccination and/or challenge. The neutralizing antibody titers were determined for plasma collected from mice in the SC-Naïve group at the times of MPXV challenge and sacrifice and for plasma collected from mice in the SC-Lister and SC-LC16m8 groups at the times of immunization with Lister or LC16m8, challenge with MPXV, and sacrifice. The day of MPXV challenge was defined as day zero.