Tiantan vaccinia virus-based vaccine with promising safety provides sustained protection against mpox in non-human primates

Abstract

The World Health Organization (WHO) has declared the mpox outbreak a public health emergency of international concern (PHEIC). Safe and efficient vaccines against the mpox virus (MPXV) are urgently needed to impede the surge in cases. Here, we report the results of a preclinical study employing different dosing strategies on a vaccine candidate named NTV, obtained via targeted gene deletion in the Tiantan strain vaccinia virus, resulting in a replication-deficient variant. Following optimisation of the NTV immunization dose and confirmation of its protective efficacy against MPXV in a mouse model, we demonstrate that a two-shot NTV regimen in macaques elicits significant neutralizing antibody and cellular immune responses, providing efficient protection against MPXV challenge. Notably, we find that a single NTV dose or long-term immunization in macaques offer effective protection against moderate or severe mpox disease by enhancing cellular immunity and rapidly evoking neutralizing antibodies. These results demonstrate the vaccine's potential for emergency use and for long-lasting protection. Safety evaluations show no adverse effects in macaques receiving triple the standard dosage in three consecutive injections. These findings highlight the potential of the NTV vaccine candidate with key advantages, including robust immunogenicity, sustained protective efficacy, and safety in preclinical settings.

Fig. 1. The immunogenicity and protective efficacy of NTV vaccine in mice.

a Schematic diagram of TTV and NTV genome. The deleted genes in NTV genome compared with TTV genome are indicated. b Schematic illustrating experiment design in mice. Immunizations were performed with different doses of NTV or a mock saline treatment via intramuscular (i.m.) route on day 0 and 28 (n = 10/group). Created in BioRender. Yang, C. (2025) https://BioRender.com/qfm3k4e. c Humoral immune responses were assessed by binding antibody to NTV (geometric mean titers, GMT; left panel) via ELISA assay or by neutralizing antibodies to VACV (middle panel) and MPXV (right panel) using 50% plaque reduction neutralization tests (PRNT₅₀) following NTV boost immunization (two-shot NTV immunization regimen). n = 10 biological replicates per group. d Schematic illustrating experiment design in mice. Immunizations with different shots of NTV or TTV via i.m. route (n = 10/group). Created in BioRender. Yang, C. (2025) https://BioRender.com/qfm3k4e. e Humoral immune responses were assessed by binding antibodies to NTV (GMT, left panel) via ELISA assay or by neutralizing antibodies to VACV (middle panel) and MPXV (right panel) via PRNT₅₀ assay following NTV prime immunization (one-shot NTV immunization regimen), NTV boost immunization (two-shot NTV immunization regimen), and TTV immunization (one-shot NTV immunization regimen). n = 10 biological replicates per group. f, g Prime-boost regimen of NTV vaccine provides protection for mice against VACV and MPXV challenge. NTV (1 × 10⁸ PFU) or saline were performed intramuscularly on day 0 and 28. Fourteen days after the NTV booster, BALB/c mice were challenged with 1 × 10⁶ TCID₅₀ of VACV WR via the intranasal (i.n.) route (f, n = 12/group), and CAST/EiJ mice were challenged with 1 × 10⁶ TCID₅₀ of MPXV clade IIb via the intraperitoneal (i.p.) route (g, n = 6/group). Created in BioRender. Yang, C. (2025) https://BioRender.com/h1at7w0. Weight loss and survival rate (n = 6/group), viral loads and titers in the lungs and spleen (n = 6/group) were detected after viral challenge. The dashed line indicates the limit of detection of the assay. Differences between the groups were evaluated using two-way ANOVA (c, g) or a two-sided unpaired Student’s t-tests (e, f). The data are presented as the means ± SEM. Source data are provided as a Source Data file.

Fig. 2. Robust humoral and cellular immune responses in NTV-vaccinated rhesus macaques.

a Schematic diagram of two-dose NTV immunization regimen, sample collection, and challenge schedule in rhesus macaques. Created in BioRender. Yang, C. (2025) https://BioRender.com/pmjfete. Immunizations were performed with different doses of NTV (1/5 dose NTV: 2 × 10⁷ PFU; 1 dose NTV: 1 × 10⁸ PFU; 2 doses NTV: 2 × 10⁸ PFU) or a mock saline treatment via intramuscular (i.m.) route on day 0 and 28, and challenge was performed with 4 × 10⁷ TCID₅₀ of MPXV via intravenous (i.v.) route on day 49 after initial immunization (n = 6/group). Specific IgG binding titers against NTV (b) or MPXV-specific antigen (c) (A35R, B6R (extracellular virion, EV proteins), H3L, M1R (mature virion, MV proteins)) at baseline (day 0), after NTV prime (day 28) and boost (day 42) immunization were determined by ELISA. n = 6 biological replicates per group. The PRNT₅₀ was determined by neutralizing antibody assay based on VACV (d) or MPXV (e) at baseline (day 0), after NTV prime (day 28) and boost (day 42) immunization. The dashed line indicates the limit of detection of the assay. n = 6 biological replicates per group. CD4⁺ and CD8⁺ T cell responses to virus VACV (f) or vaccine NTV (g) by TNF-α, IFN-γ, and IL-2 intracellular cytokine staining (ICS) assays following NTV prime (day 28) and boost (day 42) immunization. The lower and upper limits of detection are indicated where applicable. n = 6 biological replicates per group. Responses depicted are % TNF-α, IFN-γ, IL-2 single-positive or TNF-α/IFN-γ/IL-2 triple-positive CD4⁺ or CD8⁺ T cells following VACV or NTV stimulation. Boxplots represent the median (central line), interquartile range (IQR, box boundaries), whiskers extending to the minimum to maximum values. Differences between the groups were evaluated using two-way ANOVA. The data are presented as the means ± SEM (b–e) or the min to max (f, g). Source data are provided as a Source Data file.

Fig. 3. Prime-boost regimen of NTV vaccine protects rhesus macaques from MPXV challenge.

a Schematic illustrating experiment design in rhesus macaques. Created in BioRender. Yang, C. (2025) https://BioRender.com/pmjfete. b The PRNT₅₀ was determined by neutralizing antibody assay based on MPXV on days 0, 10, and 28 post MPXV challenge in different doses NTV immunization groups. n = 6 biological replicates per group. c Poxvirus skin lesion counts were plotted and compared on days 0, 4, 7, 10, 14, 21, and 28 following MPXV challenge in different doses NTV immunization groups. n = 6 biological replicates per group. d Plasma log₁₀ viral DNA copies/ml per animal were assessed on days 0, 4, 7, 10, 14, 21, and 28 following MPXV challenge in the different NTV dose immunization groups. n = 6 biological replicates per group. e Tissues samples (heart, liver, spleen, lung, kidney, brain, stomach, ileum, colon, inguinal lymph node (LN), mesenteric LN) log₁₀ viral DNA copies/μg DNA collected from euthanized rhesus macaques on days 10 and 28 following MPXV challenge in the different NTV dose immunization group were assessed via quantitative PCR. n = 3 biological replicates per group. The dashed line indicates the limit of detection of the assay. Differences between the groups were evaluated using two-way ANOVA. The data are presented as the means ± SEM. Source data are provided as a Source Data file.

Fig. 4. Prime-boost regimen of NTV vaccine provides long-lasting protection for rhesus macaques against MPXV challenge.

a Schematic diagram of the long-lasting protective efficacy of NTV vaccine against MPXV challenge in rhesus macaques (n = 4/group). Created in BioRender. Yang, C. (2025) https://BioRender.com/pmjfete. Specific IgG binding titers against NTV (b) or MPXV-specific antigen (c) (A35R, B6R (extracellular virion, EV proteins), H3L, M1R (mature virion, MV proteins)) were determined by ELISA. n = 4 biological replicates per group. d The PRNT₅₀ was determined by neutralizing antibody assay based on VACV (left panel) or MPXV (right panel). n = 4 biological replicates per group. e CD4⁺ and CD8⁺ T cell responses to vaccine NTV (upper panel) or virus VACV (lower panel) by TNF-α, IFN-γ, and IL-2 ICS assays. The lower and upper limits of detection are indicated where applicable. n = 4 biological replicates per group. Boxplots represent the median (central line), interquartile range (IQR, box boundaries), whiskers extending to the minimum to maximum values. f The PRNT₅₀ was determined by neutralizing antibody assay post MPXV challenge. n = 4 biological replicates per group. g Proportion of CD8⁺ T cell subsets in PBMCs of rhesus macaques were analyzed via flow cytometry. The percentage levels of CD8⁺ T subsets (Tn: naïve T cells, Tcm: central memory T cells, Tem: effector memory T cells, Te: terminally differentiated effector T cells) were analyzed via flow cytometry. n = 4 biological replicates per group. Boxplots represent the median (central line), interquartile range (IQR, box boundaries), whiskers extending to the minimum to maximum values. Skin lesion counts (h), and plasma log₁₀ viral DNA copies/ml (i) were assessed on indicated days following MPXV challenge in long-lasting NTV immunization (the purple lines) and saline treatment (the gray lines) groups. Solid lines indicate the median number of lesions (h) or viral loads (i) for each group, while dashed lines represent individual animal data. Differences between the groups were evaluated using one-way ANOVA (b–d) or two-way ANOVA (e–g). The data are presented as the means ± SEM (b–d, f), or the min to max (e, g). Source data are provided as a Source Data file.

Fig. 5. Single-dose regimen of NTV vaccine provides robust but incomplete protection for rhesus macaques against MPXV challenge.

a Schematic diagram of the single-dose immunization of NTV vaccine against MPXV in rhesus macaques. Created in BioRender. Yang, C. (2025) https://BioRender.com/pmjfete. Sample collection, and challenge schedule in rhesus macaques. Immunizations were performed intramuscularly (i.m.), and challenge was performed intravenously (i.v.) (n = 4/group). b Specific IgG binding titers against NTV on day 0, 14 and 28 post NTV immunization were determined by ELISA. n = 4 biological replicates per group. The PRNT₅₀ was determined by neutralizing antibody assay based on VACV (c) or MPXV (d) on days 0, 14, and 28 post NTV immunization. n = 4 biological replicates per group. e CD4⁺ and CD8⁺ T cell responses to vaccine NTV (upper panel) or virus VACV (lower panel) by TNF-α, IFN-γ, and IL-2 ICS assays on days 0, 14, and 28 post NTV immunization. The lower and upper limits of detection are indicated where applicable. Responses depicted are % TNF-α, IFN-γ, IL-2 single-positive CD4⁺ or CD8⁺ T cells following VACV or NTV stimulation. The data are presented as the min to max. n = 4 biological replicates per group. f The PRNT₅₀ was determined by neutralizing antibody assay based on MPXV on days 0, and 10 post MPXV challenge in single-dose NTV immunization and saline treatment groups. n = 4 biological replicates per group. g Poxvirus skin lesion counts were plotted and compared on days 0, 4, 7, 10, 14, 21, and 28 following MPXV challenge in single-dose NTV immunization and saline treatment groups. h Plasma log₁₀ viral DNA copies/ml were assessed on days 0, 4, 7, 10, 14, 21, and 28 following MPXV challenge in single-dose NTV and saline immunization groups. The dashed line indicates the limit of detection of the assay. Differences between the groups were evaluated using one-way ANOVA (b–d) or two-way ANOVA (e, f). The data are presented as the means ± SEM (b–d, f) or the min to max (e). Source data are provided as a Source Data file.

Fig. 6. Viral absorption kinetics, viral shedding, viral biodistribution of NTV vaccine in cynomolgus monkeys.

a Schematic diagram of immunization and sample collection, and challenge schedule in cynomolgus monkeys. Different doses of NTV were immunized via intramuscular (i.m.) route (n = 10/group). Created in BioRender. Yang, C. (2025) https://BioRender.com/ylcbdnw. b Viral absorption kinetics in cynomolgus monkeys immunized with different doses of NTV. Viral DNA loads in plasma of cynomolgus monkeys at 0 h, 2 h, 24 h, 72 h, and 168 h post prime (day 0), and boost-2 (day 42) immunization of different doses of NTV (saline, 1 dose and 3 doses). n = 10 biological replicates per group. Viral shedding in urine (c), feces (d), perioral secretions (e), perinasal secretions (f), and injection site swab (g) in cynomolgus monkeys immunized with different doses of NTV. Viral DNA loads in urine, feces, perioral secretions, perinasal secretions, and infection site swab in cynomolgus monkeys on days 0, 1, 3, 7, 43, 45, 49, 69 post prime immunization with different doses of NTV (saline, 1 dose and 3 doses). n = 10 biological replicates per group. Tissues viral DNA copies/μg DNA in cynomolgus monkeys euthanized at days 45 (h) and 70 (i) post prime immunization (dppi) of different doses of NTV (saline, 1 dose and 3 doses) were assessed. n = 5 biological replicates per group. The dashed line indicates the limit of detection of the assay. The data are presented as the means ± SEM. Source data are provided as a Source Data file.

Fig. 7. Blood pressure, oxygen saturation, hematology, cytokine expression, and histopathology of NTV vaccine in cynomolgus monkeys.

a Blood pressure in cynomolgus monkeys immunized with different doses of NTV. The mean blood pressure (MBP), systolic blood pressure (SBP), and pulse pressure (PP) in cynomolgus monkeys post prime immunization with different doses of NTV (saline, 1 dose and 3 doses) were measured by sphygmomanometer (BP-2010E). b Oxygen saturation in cynomolgus monkeys immunized with different doses of NTV. Oxygen saturation (SpO2) of cynomolgus monkeys post prime immunization with different doses of NTV (saline, 1 dose and 3 doses) were measured using pulse oximetry (CMC-60C). c Hematology in cynomolgus monkeys immunized with different doses of NTV. White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), blood platelet count (PLT), lymphocyte (Lymph), lymphocyte ratio (Lymph%, the percentage of lymphocyte count in the white blood cell count), neutrophil (Neut), neutrophil ratio (Neut%, the percentage of neutrophil count in the white blood cell count), monocyte (Mono), and monocyte ratio (Mono%, the percentage of monocyte count in the white blood cell count) in peripheral blood of cynomolgus monkeys post prime immunization with different doses of NTV (saline, 1 dose and 3 doses). d Secreted cytokine quantification in cynomolgus monkeys immunized with different doses of NTV were analyzed. TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6 in serum of cynomolgus monkeys post prime immunization with different doses of NTV (saline, 1 dose and 3 doses). n = 10 biological replicates per group (a–d). e Pathological analysis of cynomolgus monkeys with and without vaccination of 3 doses of NTV. Representative histopathology (H&E) of different tissues, brain, heart, liver, spleen, lungs, and kidneys of cynomolgus monkeys undergoing necropsy on day 45 immunization with saline or 3 doses of NTV. n = 5 biological replicates per group. Bar = 500 μm. Differences between the groups were evaluated using two-way ANOVA. The data are presented as the means ± SEM. Source data are provided as a Source Data file.