Table 1.

The production method of first-, second-, and third-generation smallpox vaccines.

Vaccine generation	Name of vaccines	The production method of vaccine	Ref
First-generation	Dryvax	A live VACV preparation made from calf lymph is called Dryvax®. Lyophilization is used to purify, concentrate, and dry the calf lymph. In government vaccine stockpiles, a more recent vaccine made in labs is replacing the antigen extracted by scraping viruses from the skin of diseased calves.	[79,80]
	Aventis Pasteur Smallpox Vaccine (APSV)	Another replication-competent VACV vaccine with a projected similar safety profile to ACAM2000® is APSV, held in the Strategic National Stockpile. Currently, research is being done on vaccination. If ACAM2000® becomes unavailable, is challenging to procure, or is contraindicated for a specific person, APSV would be made accessible under an IND or EUA for use in the case of a smallpox emergency.	[81]
Second-generation	ACAM2000	A clonally purified master seed stock of the vaccinia virus, derived from the strain of vaccinia used by the New York City Board of Health, is used to create it in cell culture. Without the intrinsic mutations associated with serial replication, the clonally purified master seed ensures a more uniform vaccine, and the cell culture restricts accidental and bacterial contamination in vaccine manufacture.	[82,83]
	Lister strain isolate of vaccinia in rabbit kidney cells (RIVM)	Thus, using rabbit kidney cells, the first second-generation Lister-based vaccine, RIVM, was created in 1960. No additional passages were carried out during the creation of this vaccine; the virus was transferred directly from the calf lymph vaccine to cells. Similar take rates and neutralizing Abs were seen with the freeze-dried vaccine and the calf lymph-derived vaccine. In clinical trials, this vaccine was used in the Netherlands and Indonesia without leading to serious side effects.	[84]
	Elstree-BN	Bavarian Nordic's (BN) Lister strain-based Elstree-BN vaccine demonstrated safety and immunogenicity in monkey and human clinical studies beginning in 2004. The embryonic cells of chickens were used in the experiment. Similarly, a vaccine was developed in Japan utilizing chicken embryo fibroblast (CEF) cells before smallpox was eliminated; although its safety profile was adequate, its effectiveness was not.	[[85], [86], [87]]
	VACV Lister/CEP	Sanofi Pasteur created a second-generation VACV vaccination by undergoing three passages in chicken embryonic primary cells using a batch of the first-generation Lister vaccine (CEP). Regarding immunogenicity and safety, Lister/CEP was comparable to the original first-generation Lister vaccination.	[84,88]
Third-generation	IMVAMUNE	The dermal vaccinia strain Ankara (chorioallantois vaccinia virus Ankara (CVA)) is used to create modified vaccinia Ankara; it was first isolated from a lesion on a horse and is now housed at the Vaccination Institution Ankara in Ankara, Turkey. Although initially developed to protect cattle against OPXVs, MVA has currently been researched as a main vaccination booster for at-risk human populations using the first generation of smallpox vaccine. Over 570 consecutive passages in primary chick embryo fibroblast cells suppressed MVA.	[89]
	LC16m8	On chorioallantois membranes, a clone of LC16 that produced small pocks, a trait associated with reduced growth capacity in mammalian tissues, was chosen to minimize the possibility of autoinoculation complications brought on by a prolonged pock reaction (CAM). In the primary rabbit kidney, the virus was passed through six more times at a low temperature before growing on CAM. Small (2–3 mm) pocks were isolated and designated LC16mO. (Lister Clone 16 medium pock size on CAM original clone). The LC16mO clones were grown on CAM and passed through three additional PRK cell passages before being chosen for small (0.5–1 mm) pocks. The last attenuated clone, designated as LC16m8 (clone 8).	[90]