Comprehensive literature review of monkeypox

Figures & data

Figure 1. General structure of the MPXV genome. The genome is made up of double-stranded linear DNA (approximately 197 kb), primarily composed of hairpin loops, some open reading frames (ORFs), and tandem repeats, while the ITRs are made up of tandem repeats, hairpin loops, and some ORFs [13]. The ends of the genome form direct repeats called ITRs, and the genome has a terminal hairpin loop (no free ends). Most of the essential genes are located in the central part of the genome, and there are ∼250 genes in the genome [14]. The upper box reveals a 625-bp deletion directly upstream of the right ITR (red box), which completely removes MPV-Z-N2R (locus 201) and truncates OMCP (MPV-Z-N3R, locus 202). The central part contains the following genes: D1R: large subunit of mRNA capping enzyme, D2R and D3R: internal structural proteins of intracellular mature virions (IMVs), D4R: viral DNA glycosylase, D5R: ATPase, D6R: subunit of early transcription protein, D7R: subunit of RNA polymerase, D8R: membrane protein of IMV, D9R, D10R, D11R: nucleotide triphosphate phosphorylate, D12R: small subunit of mRNA capping enzyme, and D13R: core protein of IMV [14].

Table 1. List of the most important ORFs in the MPXV genome and their functions.

ORF	Size	Functions
D5R	242	Zinc-binding, virulence factor and inhibition of UV-induced apoptosis
P1L	117	Secretion of virulence factor
C2L	375	Synthesis of serine protease inhibitor-like (SPI-3) and prevents cell fusion
C8L	151	Deoxyuridine triphosphatase production
C16L	439	Encoding serine/threonine protein kinase 2 and regulation of virion morphogenesis
C23R	101	Virion core DNA binding phosphoprotein
F1L	479	Poly(A) polymerase and catalytic subunit
F3L	153	dsRNA binding inhibits dsRNA-dependent protein kinase and 2-5A-synthetase
F4L	259	RNA polymerase, 30-kDa subunit and intermediate stage transcription factor
F8L	1006	DNA polymerase
Q2L	108	Virion-associated glutaredoxin
I1L	312	Virosomal protein needed for virus multiplication
I3L	269	ssDNA-binding P-protein interacts with R2 subunit of ribonucleotide reductase
I4L	771	ssDNA-binding P-protein interacts with R1 subunit of ribonucleotide reductase
I7L	423	Virion core protein, DNA topoisomerase II homologue from
G4L	124	Virion-associated glutaredoxin, required for disulphide bonds and assembly
H5R	213	Virosome-associated, late gene transcription factor, VLTF-4, Ca2^+-binding motif
E5R	785	Nucleic acid-independent nucleoside triphosphatase, required for DNA replication
A11L	891	Major virion core protein p4a
A13L	190	Virion core protein
A19R	492	DNA helicase, post replicative negative transcription elongation factor
A34L	300	DNA packaging into virion and NTP-binding motif A
A50R	554	DNA ligase

Source: Adapted from Shchelkunov et al. [9].

Table 2. List of known MPXV proteins, their encoding genes and host target proteins.

Gene	Protein	Host target proteins
Entry proteins
M1R	Protein L1 (virion membrane protein)	Probably binds to host cell entry receptors
E8L	E8L (cell surface-binding protein)	Cell surface chondroitin sulphate proteoglycans (CSPG)
H3L	H3L (envelope protein)	Cell surface heparan sulphate (HS)
Exit proteins
A38R	IEV (transmembrane phosphoprotein)	NCK adaptor protein 1 (NCK1), kinesin light chain 1 (KLC1), intersectin-1 (ITSN1) and epidermal growth factor receptor substrate 15 (EPS15)
C23R	Core phosphoprotein F17	Rapamycin-insensitive companion of mTOR (RICTOR) and regulatory-associated protein of mTOR (RPTOR)
C18L	Protein F12	Kinesin light chain 2 (KLC2)
Immunomodulatory proteins
J3R	Chemokine binding protein	CC and CXC chemokines
J2L	Cytokine response-modifying protein B	Tumour necrosis factor-alpha (TNF-α), TNF-β, CC motif chemokine ligand 28 (CCL28), CCL25, CXC motif chemokine ligand 12 (CXCL12), CXCL13 and CXCL14
D9L	Ankyrin repeat domain containing protein CP77 (type I interferon (IFN) evasion protein)	Cullin-1 (CUL1) in the SKP1-CUL1-F-Box (SCF) complex
F3L	RNA-binding protein E3	Interferon-stimulated gene 15 (ISG15), eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2)/protein kinase R (PKR) and Z-DNA binding protein 1 (ZBP1)
H1L	Dual specificity protein phosphatase H1	Signal transducer and activator of transcription 1 (STAT1)
D3R	EGFR binding protein (MPXgp006)	Epidermal growth factor receptor (EGFR)
D11L	Protein C6	TRAF family member associated NF-κB activator (TANK), TANK-binding kinase 1-binding protein 1 (TBKBP1), 5-azacytidine-induced protein 2 (AZI2) and STAT2
C7L	Protein F1	Bcl-2-like protein 11 (BCL2L11), NLR family pyrin domain containing 1 (NLRP1) and Bcl-2 homologous antagonist/killer (BAK)
B16R	Soluble IFN-alpha receptor	IFN-α
C1L	IFN antagonist K1L	IFN
B13R	Protein B13	IκB kinase β (IKKβ)
B9R	Soluble IFN-γ receptor B8	IFN-γ
P1L	Protein N1	Bcl-2-associated agonist of cell death (BAD) and Bcl-2-associated X protein (BAX)
C6R	Protein K7	DEAD-box helicase 3 X-linked (DDX3X)
A37R	MHC modulating protein	Major histocompatibility complex (MHC) class II
A41L	Protein A41 (chemokine binding protein)	CCL21, CCL25, CCL26 and CCL28
A47R	TLR inactivating protein (MPXgp157)	Myeloid differentiation factor-88 (MyD88) adaptor-like and TIR-domain containing adapter-inducing interferon-β (TRIF)-related adaptor molecule (TRAM)

Data were collected from https://viralzone.expasy.org/9976 and https://www.uniprot.org/.

Figure 2. Steps of MPXV entry into host cells [13,14,59–62]. (1) Schematic of the structure of MPXV. (2) Both the EEV and IMV virions penetrate the host membrane by binding and macropinocytosis. MPXV virions use glycosaminoglycans as host receptors. (3) After the internal virion components enter the cytoplasm, core uncoating occurs and this process leads to delivery of the MPXV genome and accessory proteins to the cytosol. (4) The released MPXV genome is used as a template for DNA replication. (5) Early viral DNA transcription followed by translation into the host ribosome occurs to encode essential proteins. Early proteins aid in DNA replication. (6) These proteins interact with host sensor proteins resulting in internal and external modulations. The major intracellular modulations include prevention of viral genome detection, induction of cell cycle arrest, apoptosis inhibition, inhibition of the antiviral system and modulation of some host cellular signalling pathways. Early proteins play essential extracellular roles as immunomodulatory agents and as growth factor-like domains that stimulate onset of mitosis in neighbouring cells. (7) Early proteins are used in production of intermediate proteins. (8) These proteins are involved in late transcription and translation processes and aid in DNA replication. (9) Late proteins are essential components for viral assembly. (10) Viral morphogenesis occurs by formation of inner tubular nucleocapsid structure folding and assembly of viral glycoproteins to generate MV virions. (11) Except those released via infected cell lysis, MV virions transit to the Golgi apparatus along microtubules for double membrane wrapping. (12) The resulting EEV virions exit the infected cell by two routes: by the actin tail assembly, which provides enough force to propel the virions out of the cell or by budding from a cellular membrane (Created with BioRender.com).

Figure 3. MPXV proteins (red) that participate in virostealth, viromimicry and virotransduction are responsible for immune evasion mechanisms of MPX infection [134–137]. In viromimicry, MPXV mimics host receptors that inhibit binding of IFN, IL-1β and TNF as well as MPXV-encoded chemokines and growth factors. In virotransduction, several antiviral pathways including IFN, NF-κB, IRF3 and apoptosis are interfered with by intracellular MPXV-encoded proteins to inhibit their functions. Virostealth is achieved with F1, an anti-apoptotic host range protein that helps with viral replication and the spread of MPX infection (Created with BioRender.com).

Figure 4. Common symptoms of MPX according to the WHO [160] (Created with BioRender.com).

Figure 5. Transmission routes associated with MPXV according to the WHO [160] (Created with BioRender.com).

Table 3. Number of MPX cases and deaths from 1970 to 2018.

Country	Time frame	Total suspected cases	Total deaths	References
Democratic Republic of the Congo	1970	1	1	[1]
	1981–1986	338	33	[179]
	1996–1997	773	8	[216]
	2001	388	13	[196]
	2002	881	14	[196]
	2003	755	16	[196]
	2004	1024	29	[196]
	2005	1708	26	[196]
	2006	783	20	[196]
	2007	970	11	[196]
	2008	1599	67	[196]
	2009	1919	27	[196]
	2010	2322	26	[196]
	2011	2208	15	[196]
	2012	2629	34	[196]
	2013	2460	37	[196]
	2016	155	11	[217]
	2019	3794	73	[218]
	2020	4594	171	[218]
Central African Republic	2001	8	2	[219,220]
	2010	2	0	[219]
	2015	3	1	[220]
	2015–2016	62	5	[221,222]
	2017	8	0	[193]
	2018	33	1	[223]
Republic of the Congo	2003	12	1	[224]
	2010	11	1	[225]
	2017	88	6	[193]
Sudan	2005	37	0	[226]
Cameroon	1989	1	0	[227]
	2018	16	0	[223]
Gabon	1987	1	1	[228]
	1991	9	0	[193]
Nigeria	1971	2	0	[177]
	1978	1	0	[177]
	2017–2018	228	6	[65]
Sierra Leone	1970–1971	1	0	[177]
	2014	1	1	[193]
	2017	1	0	[193]
Liberia	1970–1971	4	0	[177]
Côte d’Ivoire	1971	1	0	[229]
USA	2003	47	0	[230,231]
	2021	2	0	[232]
UK	2018	4	0	[233]
	2019	1	0	[234]
	2021	3	0	[235]
Singapore	2019	1	0	[236]

Source: Adopted from Brown and Leggat [162]; Beer and Rao [215]; Adegboye et al. [162,215,237].

Table 4. MPX cases and deaths reported by the WHO during the multi-country 2022 outbreak (as of 8 June 2022) [232].

WHO Region	Country	Confirmed cases	Suspected cases	Deaths
AFRO	Cameroon	3	28	2
	Central African Republic	8	17	2
	Republic of Congo	2	7	3
	DRC	10	1356	64
	Liberia	0	4	0
	Sierra Leone	0	2	0
	Nigeria	31	110	1
	Ghana	5	12	0
AMRO	Argentina	2	0	0
	Canada	110	0	0
	Mexico	1	0	0
	United States of America	40	0	0
EMRO	United Arab Emirates	13	0	0
	Morocco	1	0	0
EURO	Austria	1	0	0
	Belgium	24	0	0
	Czech Republic	6	0	0
	Denmark	3	0	0
	Finland	3	0	0
	France	66	0	0
	Germany	113	0	0
	Hungary	2	0	0
	Ireland	9	0	0
	Italy	29	0	0
	Israel	2	0	0
	Latvia	2	0	0
	Malta	1	0	0
	Netherlands	54	0	0
	Norway	2	0	0
	Portugal	191	0	0
	Slovenia	6	0	0
	Spain	259	0	0
	Sweden	6	0	0
	Switzerland	12	0	0
	The United Kingdom	321	0	0
WPRO	Australia	6	1	0
Cumulative	36 countries	1344	1537	72

AFRO, Africa; AMRO, Americas; EMRO, Eastern Mediterranean, EURO, Europe; WPRO, Western Pacific; The DRC, Democratic Republic of the Congo

Figure 6. Histology and negative-stain electron microscopic views of MPXV virions (Created with BioRender.com).

Table 5. List of clinical trials of drugs and vaccines against MPX.

Product	Identifier	Type	Developer, Country	Trial location	Status	First Posted
IMVAMUNE®	NCT02977715	SPX vaccine (attenuated live virus)	Bavarian Nordic, Denmark	DRC	Active, not recruiting	30 November 2016
Imvamex®	NCT03745131	SPX vaccine (attenuated live virus)	Bavarian Nordic, Denmark	United Kingdom	Completed	19 November 2018
ST-246®	NCT00728689	Anti-orthopoxvirus compound inhibits release of extracellular virus by targeting the F13L W protein	SIGA Technologies, Inc., United States	United States	Completed	6 August 2008
Tecovirimat (TPOXX, previously ST-246)	NCT02080767	Anti-orthopoxvirus drug that interferes with a p37 viral protein	SIGA Technologies, Inc., United States	-	Available (FDA-approved)	6 March 2014

Data were collected from https://clinicaltrials.gov/.

ST-246®, 4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop [f]isoindol-2(1 H)-yl)-benzamide; DRC, Democratic Republic of the Congo.

Table 6. Promising anti-MPX medications.

Name	Structure and nature	Evidence	Other Uses and notes	Combined therapy	Side effects
Cidofovir, also called HPMPC or Vistide [269]	[(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] [266] acyclic cytosine phosphonate analogue [272]	Cidofovir has been successfully used in humans to treat persistent molluscum contagiosum and orf in immunocompromised individuals using topical and intravenous routes [266] Targets the viral DNA polymerase and inhibits poxvirus replication in-vitro, ex-vivo and in vivo [272] Effective against almost all DNA viruses and has a broad spectrum of activities [266–268] Starting antiviral treatment 24 h after deadly intratracheal MPX infection using either of the antiviral drugs and a variety of systemic treatment regimens resulted in significantly lower mortality and cutaneous MPX lesions. All surviving animals had virus-specific blood antibodies and antiviral T cells after the antiviral medication was stopped 13 days after infection [269] Three non-human primates were pox-antigen negative after exposure to a fatal dose of MPX and receiving cidofovir. All three primates survived [273]	Resistant orthopox viruses were thought to be untreatable with cidofovir; however, their virulence might be reduced [274] When given as early as 5 days before infection or as late as 3 days after infection with either CPXV or VACV, a single dose pre- or post-treatment with cidofovir at 3–100 mg/kg in mouse models proved successful [271]	Coadministration of cidofovir and Dryvax in a single dosage efficiently minimized vaccination side effects against MPX but severely compromised vaccine-elicited immunological responses and vaccine-induced immunity [275]	Exposure to cidofovir that will have a protective effect against certain orthopox viruses (i.e. VARV major virus) will need doses that are 5–10 times that currently administered to humans, such doses may lead to nephrotoxicity [267]
HPMPA	((S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine) [264]	HPMPA was effective against several orthopoxviruses, with IC50s of 10.9 and 9.2 μg/ml for MPX Vero and LLC-MK2 infected cells, respectively [264] In vitro, (S)-HPMPA is active against numerous orthopoxviruses, but not in vivo. Ether lipid esters, such as ODE-(S)-HPMPA and HDP-(S)-HPMPA, exhibit better bioavailability and activity against CPXV and VACV infections in mice [276]	HPMPA is closely linked to cidofovir and works similarly to prevent viral replication. Because cidofovir has already been approved for use in the US and is far more likely to be used to treat poxvirus infections, HPMPA is unlikely to be used in US clinical trials [264]		Oral HDP-(S)-HPMPA and ODE-(S)-HPMPA showed no effect. ODE-(S)-HPMPA, given 72 h after CPXV infection or 24 or 48 h after VACV infection, also reduced mortality. Both compounds have the potential to cure human cases of SPX vaccination-related adverse effects [276]
Brincidofovir (CMX001) HDP-cidofovir	Lipophilic nucleotide analogue formed by covalently linking 3-(hexdecyloxy)propan-1-ol to cidofovir (hexadecyloxypropyl ester of cidofovir) [277]	In comparison to CDV, CMX001 has significantly improved efficacy against all dsDNA viruses [278] Antiviral treatment with CMX001 and ST-246 protects mice infected with the murine MPX in a STAT1-deficient C57BL/6 mouse model [279] CMX001 is effective for symptomatic RPV infection (an orthopoxvirus infection) [280]	Orally available [278]	When administered together on the day of infection, CMX001 and ST-246 provide protection [279]	To date, there has been no evidence of nephrotoxicity in healthy volunteers or critically ill transplant patients [278]
Tecovirimat (ST-246) [281]		Tecovirimat is a new antiviral that targets viral p37 protein orthologs to prevent orthopoxviruses from egressing [281] Has antiviral activity in a variety of animal models [272]	Tecovirimat works as an antiviral by suppressing the generation of EVs, which prevents cell-cell and long-distance propagation [282]	ACAM2000 (SPX vaccine) given after exposure did not protect against severe MPX disease or death, while post-exposure treatment with tecovirimat alone or with ACAM2000 gave complete protection. Tecovirimat after infection was 83% (days 4 and 5) or 50% (day 6) effective [261]	Following a phase I clinical investigation, safety was validated in healthy human volunteers [272]
Retro-2 and analogues		PA104 was the most effective Retro-2 analogue, suppressing viral propagation by 90% at 1.3 M with a good selectivity index. These findings, and additional identification of PA104’s specific protein targets and in-vivo activity, could be significant for development of effective antivirals for OPXV [282]			PA104 inhibited two different ST-246-resistant viruses, indicating that it could be useful in combination therapy with ST-246 [282]
siRNAs		At a dose of 1 nM, siRNAs siB1R-2 and siG7L-1 reduced MPXV viral proliferation by 95%. Without inducing a beta interferon response, siB1R-2 and siG7L-1 silenced their corresponding transcript: B1R and G7L mRNAs [283] Seven siRNA constructs suppressed viral replication in cell culture by 65-95% with no apparent cytotoxicity, targeting either an essential gene for viral replication (A6R) or a key gene in viral entry (E8L). Further tests using wild-type and recombinant MPX producing green fluorescent protein revealed that siA6-a was the most powerful construct, inhibiting viral replication for up to 7 days at a dose of 10 nM [284] Different pathogenic orthopoxviruses (CPXV and MPXV) were inhibited up to 70% at the lowest concentration (1 nM) tested, indicating siD5R-2 (siRNA targeting D5 protein) efficacy. siD5R-2 had antiviral effects in human keratinocyte and fibroblast cell cultures infected with VACV [285]		When siB1R-2, siG7L-1, or siD5R-2 were coupled with cidofovir, strong synergistic effects were seen. Combination therapy of siRNA and cidofovir could be effective in treating poxvirus infections [283]
Mycophenolic acid (MPA)		In plaque reduction experiments, MPA suppressed CMPV, CPXV, MPXV and VACV by 50% in African green monkey kidney (Vero 76) and mouse 3T3 cells at 0.2–3 μM [286]	Anti-orthopoxvirus efficacy of ribavirin is boosted by other modes of virus inhibition. Biological variations in mode of action and the immunosuppressive potential of ribavirin and MPA, cause the former to be effective against orthopoxvirus infections in animals, while the latter is not [286]		MPA and ribavirin were more toxic to replicating cells than stationary cell monolayers, with greater toxicity in 3T3 cells than Vero 76 cells. Compared to Vero 76 cells, the higher antiviral efficacy and toxicity of ribavirin were due to more accumulation of mono-, di- and triphosphate forms of the drug in 3T3 cells. Virus inhibition was linked to suppression of intracellular guanosine triphosphate pools for MPA and ribavirin [286]
Ribavirin		Ribavirin was significantly more effective against these viruses in 3T3 cells (IC50 2–12 μM) than in Vero 76 cells (IC50 30–250 μM) [286]

Table 7. Vaccines of various sorts and their potential applications in the prevention and treatment of MPX.

Vaccine	Efficacy against MPX	Notes
First-generation SPX vaccines: Dryvax and live VACV strains like Lister, Copenhagen (Cop), chorioallantois VACV Ankara (CVA), Tian Tan (TT), Bern and New York City Board of Health (NYCBH)	– Dryvax (freeze-dried vaccine) protects against SPX and MPX [292] – Due to a high degree of sequence conservation, vaccinating against SPX with VACV may protects against MPX [290]	– Associated with serious complications in both naïve and immune people [293]. First-generation SPX vaccines were used in the eradication program. These VACV strains caused varying levels of vaccine-related complications and as a result, strains like Bern and Copenhagen were favoured less than Lister and NYCBH [294] – Coadministration of cidofovir and Dryvax in a single dose greatly reduced adverse effects, but severely compromised vaccine-elicited immunological responses and vaccine-induced immunity to MPX [275] – Contraindicated in immunocompromised people [292]
VACV vaccine strain LC16m8	– Although lesions were smaller than those induced by the original Lister strain, LC16m8 is less attenuated than MVA and retains the potential to multiply and cause lesions in human vaccinees [295] – Due to a frameshift mutation, LC16m8 does not express B5R protein [296]. As a result, a tiny plaque phenotype develops. Humans immunized with LC16m8 have inadequate immune responses to this protein [297], which is a key target for antibodies that neutralize the EEV form of VACV [298] – Efficacy was compared to that of the original Lister strain. Monkeys were immunized with LC16m8 or Lister and subsequently infected with MPXV strain Liberia or Zr-599 intranasally or subcutaneously. With intranasal-inoculation, immunized monkeys had no symptoms of MPX, but nonimmunized controls showed normal symptoms. With subcutaneous injection, monkeys immunized with LC16m8 showed no signs of MPX except for a small ulcer at the site of MPXV inoculation, while nonimmunized controls showed fatal and typical symptoms. These findings imply that LC16m8 protects monkeys from deadly MPX and may elicit protective immunity against SPX [299]	– Derived from Lister strain in Japan [294] and harbours a mutation in the critical membrane protein B5R [299]
Modified VACV Ankara (MVA); also known as Imvanex in the European Union, Imvamune in Canada, and Jynneos in the United States [300]	– In a STAT1-deficient C57BL/6 mouse model of MPX, vaccination with MVA followed by a booster vaccine protect against an intranasal MPX challenge and elicits a more robust immune response than a single vaccination [279] – In humans and immunocompromised animals, it is safe. The MVA-based SPX vaccine protected macaques against a deadly respiratory challenge of MPX, making it a promising contender for human protection [293] – To produce immune responses comparable to those induced by the initial SPX vaccines, higher viral titres and multiple doses are required [301–303] – Before MPX viral challenge, there was no significant difference in neutralizing antibody levels in animals vaccinated with a single ACAM2000 immunization (132 U/ml) vs. a prime-boost Imvamune regimen (69 U/ml) [304]	– A highly attenuated replication-deficient strain of VV [293] derived from strain CVA by Mayr and colleagues in Germany [305] – Obtained after passages of the CVA strain in chicken embryo fibroblasts (CEFs), roughly 15% of its genome was lost due to six big deletions and many smaller alterations [306,307]. MVA is unable to reproduce in most mammalian cells in culture, including human cells and lacks several immunomodulators and host range genes seen in other VACV strains [308,309] – The prime-boost Imvamune group had evidence of viral excretion from the throats of two of six animals after challenge [304]
New York VACV (NYVAC)	NYVAC, like MVA, is unable to replicate in human cells. However, unlike MVA, expression of several late NYVAC proteins is restricted due to a translational block [310]. Despite being attenuated, NYVAC still elicits a strong immunological response [311,312]	Derived from a plaque-cloned isolate of the Copenhagen vaccine strain by deletion of 18 ORFs from the viral genome. Among the deleted ORFs, two genes are involved in nucleotide metabolism, the thymidine kinase (ORF J2R) and the large subunit of the ribonucleotide reductase (ORF I4L) [313]
DNA vaccine consisting of four VACV genes (L1R, A27L, A33R and B5R)	– After an otherwise deadly challenge with MPX, rhesus macaques treated with a DNA vaccine were protected from severe illness. Vaccinated animals with a single gene (L1R) that encodes a neutralizing antibody target developed severe illness but survived. This is the first proof that vaccinating against SPX and MPX with a subunit vaccine is possible [289] – Animals given only DNA did not have high titre Abs, had numerous skin lesions after being challenged and died similarly to placebo controls. Animals given proteins had moderate to severe illness (20-155 skin lesions) but lived. Individuals inoculated with DNA and then boosted with proteins had minimal illness, with 15 or fewer lesions that resolved in a matter of days [292]
Dryvax-derived ACAM2000 (second-generation smallpox vaccines)	– ACAM2000 did not provide protection against severe MPX disease or mortality when given after exposure [261] – Prairie dogs were protected to some extent from the 2 LD 50 challenge of MPX disease, but not from the 170 LD 5 challenge. In the 2 LD 50 challenge, giving the vaccine one day after exposure was more effective than giving it three days later for Imvamune, while ACAM2000 was equally efficacious at both post-exposure vaccination times [314]	– Treatment with tecovirimat alone or in combination with ACAM2000 after exposure gave complete protection [261] – The only SPX vaccine now available in the United States that has been licensed by the FDA [294]
– Elstree-RIVM and Elstree-BN	No substantial reduction in mortality was seen in monkeys vaccinated 24 h after MPX infection with a typical human dose of Elstree-RIVM. All surviving animals had virus-specific blood antibodies and antiviral T cells after antiviral medication was stopped 13 days after infection [269]	– Elstree-RIVM is a first-generation SPX vaccine produced on calf skins [293] – Elstree-BN is a second-generation vaccine, passaged and produced on CEFs to further attenuate the virus and to make a better-defined vaccine preparation that does not depend on the use of calves [293]

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