Evaluation of the immunobiological properties of the attenuated strain CP-65 K for the development of a cowpox vaccine

Dias Muzarap, Anar Dzhaldybayeva, Muratbay Mambetaliyev, Yekaterina Ostapchuk, Moldir S. Tuyskanova, Shalkhar Tabys, N. A. Sarsenkulova, Aisulu Valiyeva, Marzhan Kenzhebayeva, Nurshat Abdolla

8 more authors

Sanat Kilibayev, Moldir Azanbekova, Nurbek Aldayarov, Balzhan Sh. Myrzakhmetova, Кутумбетов Леспек Бекболатович, Аралбек Рсалиев, Yergali O. Abduraimov, Kuandyk D. Zhugunissov

2026

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DOI 10.1016/j.vaccine.2026.129239
Data confidence 70 medium
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Abstract

This study evaluated the suitability and immunobiological characteristics of the attenuated strain CP-65 K for the development of a cowpox vaccine. The attenuated strain CP-65 K was non-reactogenic in both laboratory and target animals and demonstrated higher immunogenicity than vaccinia virus (VACV) strains RIBSP-B and RIBSP-V, as well as the camelpox virus (CMLV) strain KM-40, supporting its potential as a live vaccine candidate. After 15 serial passages in lamb kidney cell culture, CP-65 K retained its infectious titer (6.50 ± 0.14 to 6.67 ± 0.08 log₁₀ TCID₅₀/mL) and induced specific antibodies in vaccinated rabbits by day 21 post-immunization, confirming phenotypic stability. Administration of vaccine doses ranging from 3162 to 316,200 TCID₅₀ to laboratory animals caused no clinically significant adverse effects or mortality. Subcutaneous immunization with 10,000 TCID₅₀ induced a strong humoral immune response, with neutralizing antibody titers ranging from 1:16 to 1:64 in the virus neutralization test (VNT) and enzyme-linked immunosorbent assay (ELISA) optical density values of 1.031–1.328. In vaccinated cattle, antigen-specific antibodies against cowpox virus (CPXV) were detected from day 14 after primary vaccination and increased following booster immunization. However, no statistically significant activation of T-cell responses, including changes in CD4 + /CD8 + lymphocyte populations or expression of TNF-α, IFN-γ, and IL-4 after in vitro CPXV stimulation, was observed. Challenge infection with the epizootic strain Cowpox-CAM on day 30 post-vaccination did not induce clinical signs of cowpox in vaccinated animals, whereas control animals developed hyperthermia, characteristic skin lesions, local necrosis, and evidence of systemic infection. Histological analysis confirmed pronounced local dermal lesions in non-vaccinated cattle. Antigen-specific lymphocyte proliferation observed in vitro was not accompanied by statistically significant changes in proliferating CD4 + or CD8 + T-cell subsets, and the cellular origin of the proliferating population requires further phenotypic characterization.