1 **Annex 16. Item 5.1. – Chapter 3.9.1. African swine fever (infection with African swine fever virus)**

###### 2 MEETING OF THE BIOLOGICAL STANDARDS COMMISSION

3 **Paris, 4–8 September 2023**

4

5	**SEC T I O N  3. 9.**

# 6	S U I D A E

**USA COMMENTS IN RED FONT**

7	**CHAP T E R  3 . 9 . 1 .**

# 8	A F R I C A N S WI N E F EV E R

9	**( I N F E C T I ON WI T H A F R I CA N SWI N E FEVER VI R US)**

10	**SUMMARY**

11 *African swine fever*(*ASF*) *is an infectious disease of domestic and wild pigs of all breeds and ages, caused*

12 *by ASF virus*(*ASFV*)*. The clinical syndromes vary from peracute, acute, subacute to chronic, depending on*

13 *the virulence of the virus. Acute disease is characterised by high fever, haemorrhages in the*

14 *reticuloendothelial system, and a high mortality rate. Soft ticks of the*Ornithodoros *genus, especially*O.

15 moubata *and*O. erraticus*, have been shown to be both reservoirs and transmission vectors of ASFV. The*

16 *virus is present in tick salivary glands and passed to new hosts*(*domestic or wild suids*) *when feeding. It can*

17 *be transmitted sexually between ticks, transovarially to the eggs, or transtadially throughout the tick’s life. In an outbreak, animal-to-animal transmission (direct contact with infected pigs or their fluids) is an important method of spread.*

18 **RATIONALE:**In a study under field conditions, 50% of the pigs in the comingled contact group developed ASFV-specific antibodies. This appears to confirm a certain level of contact transmission. https://www.mdpi.com/1999-4915/14/5/896.

19 

20 *ASFV is the only member of the*Asfarviridae *family, genus*Asfivirus*.*

21 *Laboratory diagnostic procedures for ASF fall into two groups: detection of the virus and serology. The*

22 *selection of the tests to be carried out depends on the disease situation and laboratory diagnostic capacity*

23 *in the area or country.*

24 ***Identification of the agent:****Laboratory diagnosis must be directed towards isolation of the virus by*

25 *inoculation of pig leukocyte or bone marrow cultures, the detection of antigen in smears or cryostat sections*

26 *of tissues by fluorescent antibody test and/or the detection of genomic DNA by the polymerase chain reaction*

27 (*PCR*) *or real-time PCR. The PCRs are excellent, highly sensitive, specific and rapid techniques for ASFV*

28 *detection and are very useful under a wide range of circumstances. They are especially useful if the tissues*

29 *are unsuitable for virus isolation and antigen detection. In doubtful cases, the material is passaged in*

30 *leukocyte cell cultures and the procedures described above are repeated.*

31 ***Serological tests:****Pigs that survive natural infection usually develop antibodies against ASFV from 7–10*

32 *days post-infection and these antibodies persist for long periods of time. Where the disease is endemic, or*

33 *where a primary outbreak is caused by a strain of low or moderate virulence, the investigation of new*

34 *outbreaks should include the detection of specific antibodies in serum or extracts of the tissues submitted.*

35 *A variety of methods such as the enzyme-linked immunosorbent assay*(*ELISA*)*, the indirect fluorescent*

36 *antibody test*(*IFAT*)*, the indirect immunoperoxidase test*(*IPT*), *and the immunoblotting test*(*IBT*) *is available*

37 *for antibody detection.*

38 ***Requirements for vaccines****: ~~At present, there is no vaccine for ASF.~~Commercially produced modified live*

39 *virus vaccines are available and licenced in some countries.*

## 38	A. INTRODUCTION

39 The current distribution of African swine fever (ASF) extends across more than 50 countries in three continents (Africa,

40 Asia and Europe). Several incursions of ASF out of Africa were reported between the 1960s and 1970s. In 2007, ASF was

41 introduced into Georgia, from where it spread to neighbouring countries including the Russian Federation. From there ASF

42 spread to eastern European countries extending westwards and reaching the European Union in 2014. Further westward

43 and southern spread in Europe has occurred since that time. In all these countries, both hosts – domestic pig and wild

44 boar – were affected by the disease. In August 2018, the People’s Republic of China reported its first outbreak of ASF and

45 further spread in Asia has occurred.

46 ASF virus (ASFV) is a complex large, enveloped DNA virus with icosahedral morphology. It is currently classified as the

47 only member of the *Asfaviridae*family, genus *Asfivirus*(Dixon *et al.*, 2005). More than 60 structural proteins have been

48 identified in intracellular virus particles (200 nm) (Alejo *et al.,*2018). More than a hundred infection-associated proteins

49 have been identified in infected porcine macrophages, and at least 50 of them react with sera from infected or recovered

50 pigs (Sánchez-Vizcaíno & Arias, 2012). The ASFV double-stranded linear DNA genome comprises between 170 and

51 193 kilobases (kb) and contains between 150 and 167 open reading frames with a conserved central region of about 125

52 kb and variable ends. These variable regions encode five multigene families that contribute to the variability of the virus

53 genome. The complete genomes of several ASFV strains have been sequenced (Bishop *et al.,*2015; Chapman *et al.,*

54 2011; de Villiers *et al.,*2010; Portugal *et al.,*2015). Different strains of ASFV vary in their ability to cause disease, but at

55 present there is only one recognised serotype of the virus detectable by antibody tests.

56 The molecular epidemiology of the disease is investigated by sequencing of the 3’ terminal end of the B646L open reading

57 frame encoding the p72 protein major capsid protein, which differentiates up to 24 distinct genotypes (Achenbach *et al*.,

58 2017; Boshoff *et al.,*2007; Quembo *et al.*2018). To distinguish subgroups among closely related ASFV, sequence analysis

59 of the tandem repeat sequences (TRS), located in the central variable region (CVR) within the B602L gene (Gallardo *et*

60 *al.,*2009; Lubisi *et al.,*2005; Nix *et al.,*2006) and in the intergenic region between the I73R and I329L genes, at the right

61 end of the genome (Gallardo *et al.,*2014), is undertaken. Several other gene regions such as the E183L encoding p54

62 protein, the CP204L encoding p30 protein, and the protein encoded by the EP402R gene (CD2v), have been proved as

63 useful tools to analyse ASFVs from different locations and hence track virus spread.

64 ASF viruses produce a range of syndromes varying from peracute, acute to chronic disease and subclinical infections.

65 Pigs are the only domestic animal species that is naturally infected by ASFV. European wild boar and feral pigs are also

66 susceptible to the disease, exhibiting clinical signs and mortality rates similar to those observed in domestic pigs. In contrast

67 African wild pigs such as warthogs (*Phacochoerus aethiopicus*), bush pigs (*Potamochoerus porcus*) and giant forest hogs

68 (*Hylochoerus meinertzhageni*) are resistant to the disease and show few or no clinical signs. These species of wild pig act

69 as reservoir hosts of ASFV in Africa (Costard *et al.,*2013; Sánchez-Vizcaíno *et al.,*2015).

70 The incubation period is usually 4–19 days. The more virulent strains produce peracute or acute haemorrhagic disease

71 characterised by high fever, loss of appetite, haemorrhages in the skin and internal organs, and death in 4–10 days,

72 sometimes even before the first clinical signs are observed. Case fatality rates may be as high as 100%. Less virulent

73 strains produce mild clinical signs – slight fever, reduced appetite and depression – which can be readily confused with

74 many other conditions in pigs and may not lead to suspicion of ASF. Moderately virulent strains are recognised that induce

75 variable disease forms, ranging from acute to subacute. Low virulence, non-haemadsorbing strains can produce subclinical

76 non-haemorrhagic infection and seroconversion, but some animals may develop discrete lesions in the lungs or on the

77 skin in areas over bony protrusions and other areas subject to trauma. Animals that have recovered from either acute,

78 subacute or chronic infections may potentially become persistently infected, acting as virus carriers. The biological basis

79 for the persistence of ASFV is still not well understood, nor it is clear what role persistence plays in the epidemiology of

80 the disease.

81 ASF cannot be differentiated from classical swine fever (CSF) by either clinical or post-mortem examination, and both

82 diseases should be considered in the differential diagnosis of any acute febrile haemorrhagic syndrome of pigs. Bacterial

83 septicaemias may also be confused with ASF and CSF. Laboratory tests are essential to distinguish between these

84 diseases.

85 In countries free from ASF but suspecting its presence, the laboratory diagnosis must be directed towards isolation of the

86 virus by the inoculation of pig leukocyte or bone marrow cultures, the detection of genomic DNA by polymerase chain

87 reaction (PCR) or the detection of antigen in smears or cryostat sections of tissues by direct fluorescent antibody test

88 (FAT). Currently the PCR is the most sensitive technique and can detect ASFV DNA from a very early stage of infection in

89 tissues, ethylene diamine tetra-acetic acid (EDTA)-blood and serum samples. The PCR is particularly useful if samples

90 submitted are unsuitable for virus isolation and antigen detection because they have undergone putrefaction. Pigs that

91 have recovered from acute, subacute or chronic infections usually exhibit a viraemia for several weeks making the PCR

92 test a very useful tool for the detection of ASFV DNA in pigs infected with low or moderately virulent strains. Virus isolation

93 by the inoculation of pig leukocyte or bone marrow cultures and identification by haemadsorption tests (HAD) are

94 recommended as a confirmatory test when ASF is positive by other methods, particularly in the event of a primary outbreak

95 or a case of ASF.

96 ~~As no vaccine is available, the presence of ASFV antibodies is indicative of previous infection and, as antibodies are~~

97 ~~produced from the first week of infection and persist for long periods, they are a good marker for the diagnosis of the~~

98 ~~disease, particularly in subacute and chronic forms.~~

99 Vaccines should be prepared in accordance with Chapter 1.1.8 *Principles of veterinary vaccine production*. ASF modified

100 live virus (MLVs) vaccines are based on the live virus that have been naturally attenuated or attenuated by targeted genetic

101 recombination through cell cultures (Gladue & Borca, 2022). MLV production is based on a seed-lot system consistent with

102 the *European Pharmacopoeia*(11th edition) and that has been validated with respect to virus identity, sterility, purity,

103 potency, safety, non-transmissibility, stability and immunogenicity. ASF MLV first generation vaccines – defined as those

104 for which peer-reviewed publications are in the public domain – should meet or exceed the minimum standards as

105 described below. Paramount demonstration of acceptable safety and efficacy against the epidemiologically relevant ASFV

106 field strain(s) where the vaccine is intended for use are required. At the present time, acceptable efficacy should be shown

107 against the B646L (p72) genotype II pandemic virus lineage currently circulating widely in domestic pigs and wild boar.

108 ASF MLV first generation vaccines allowing the differentiation of infected animals from vaccinated animals (DIVA) by

109 suitable methods (e.g. serology-based tests) are preferred. Demonstration of MLV safety and efficacy in breeding-age

110 boars, gilts and pregnant sows, and onset and duration of protective immunity, are also preferred but are not required to

111 meet the minimum standard.

112 ASF epidemiology is complex with different epidemiological patterns of infection occurring in Africa and Europe. ASF

113 occurs through transmission cycles involving domestic pigs, wild boar, wild African suids, and soft ticks (Sánchez-Vizcaíno

114 *et al.,*2015). In regions where *Ornithodoros*soft-bodied ticks are present, the detection of ASFV in these reservoirs of

115 infection contributes to a better understanding of the epidemiology of the disease. This is of major importance in

116 establishing effective control and eradication programmes (Costard *et al*., 2013).

117 ASF is not a zoonotic disease and does not affect public health (Sánchez-Vizcaíno *et al.*, 2009).

118 ASFV should be handled with an appropriate level of bio-containment, determined by risk analysis in accordance with

119 Chapter 1.1.4 *Biosafety and biosecurity: Standard for managing biological risk in the veterinary laboratory and animal*

120 *facilities*.

121	. . .

## 122	C. REQUIREMENTS FOR VACCINES

123 ~~At present there is no commercially available vaccine for ASF.~~

124 **1.	Background**

125 The ASF p72 genotype II strain (ASFV Georgia 2007/1 lineage) (NCBI, 2020) is recognised to be the current highest global

126 threat for domestic pig production worldwide (Penrith *et al.,*2022).

127 Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 *Principles of Veterinary Vaccine Production*.

128 Varying additional requirements relating to quality (including purity and potency), safety, and efficacy will apply in particular

129 countries or regions for manufacturers to comply with local regulatory requirements.

130 Wherever live, virulent ASFV or ASF MLVs are stored, handled and disposed, the appropriate biosecurity level, procedures

131 and practices should be used. The ASF MLV vaccine production facility should meet the requirements for containment

132 outlined in Chapter 1.1.4 *Biosafety and biosecurity: Standard for managing biological risk in the veterinary laboratory and*

133 *animal facilities*.

134 An optimal ASF MLV first generation vaccine for the target host should have the following general characteristics (minimum

135 standards):

136 	Safe: demonstrate absence of fever and clinical signs of acute or chronic ASF in vaccinated and in-contact animals,

137 minimal and ideally no vaccine virus transmission, and absence of an increase in virulence (genetic and phenotypic

138 stability);

139 	Efficacious: protects against mortality, reduces acute disease (fever accompanied by the appearance of clinical signs

140 caused by ASF) and reduces vertical (boar semen and placental) and horizontal disease transmission;

141 	Quality – purity: free from wild-type ASFV and extraneous microorganisms that could adversely affect the safety,

142 potency or efficacy of the product;

143 	Quality – potent: the log10 virus titre maintained throughout the vaccine shelf life that guarantees the efficacy

144 demonstrated by the established minimum immunising (protective) dose.

145 	Identity: based on the capacity to protect against the ASFV B646L (p72) genotype II pandemic strain or other p72

146 genotypes of recognised epidemiologic importance.

147 Vaccine production should be carried out using a validated, controlled and consistent manufacturing process.

148 ASF MLV first generation vaccines must be safe (i.e. an acceptable safety profile) for non-target species and the

149 environment in general.

150 Ideally, ASF MLV first generation vaccines that meet the minimum standards should also fulfil the following additional

151 general characteristics: i) prevents acute and persistent (carrier state) disease; ii) prevents horizontal and vertical disease

152 transmission; iii) induces rapid protective immunity (e.g. < 2 weeks); and iv) confers stable, life-long immunity.

153 Furthermore, ASF MLV second and future generation vaccines should meet the minimum safety and efficacy standards

154 as ASF MLV first generation vaccines, and ideally provide additional product profile benefits, including but not limited to: i)

155 contain a negative marker allowing the differentiation of infected from vaccinated animals (DIVA) by reliable discriminatory

156 tests such as serology-based tests; and ii) confer broad range of protection against other p72 genotype field strains of

157 varying virulence (low, moderate, and high).

158 The majority of ASF global vaccine research groups and companies are currently focused on ASF MLV first generation

159 vaccine candidates that are safe and efficacious against ASF viruses belonging to the ASFV p72 genotype II pandemic

160 strain (ASFV Georgia 2007/1 lineage) (NCBI, 2020).

161 Currently, two gene deleted MLV recombinant vaccines (ASFV-G-ΔI177L and ASFV-G-ΔMGF) have been licenced for

162 field use in Vietnam following supervised field testing to evaluate the safety and effectiveness of several vaccine batches.

163 There are numerous, promising ASF MLV vaccine candidates targeting the p72 genotype II pandemic strain under

164 development, including:

165 	A naturally attenuated field strain (Lv17/WB/Rei1) (Barasona *et al.,*2019) being developed as an oral bait vaccine for

166 wild boars;

167 	A laboratory thermo-attenuated field strain (ASFV-989) (Bourry *et al.,*2022);

168 	Single gene-deleted, recombinant viruses (e.g. SY18ΔI226R, ASFV-G-ΔA137R) (Gladue *et al*., 2021; Zhang *et al*.,

169	2021);

1 	Double gene-deleted, recombinant viruses (e.g. ASFV-G-Δ9GL/ΔUK; ASFV-SY18-∆CD2v/UK; Arm-ΔCD2v-ΔA238L)

2 (O’Donnell *et al*., 2016; Pérez-Núñez *et al*., 2022; Teklue *et al*., 2020);

3 	Multiple gene-deleted, recombinant viruses (ASFV-G-ΔI177L/ΔLVR; ASFV-G-ΔMGF; BA71ΔCD2; HLJ/18-7GD;

4 ASFVGZΔI177LΔCD2vΔMGF) (Borca *et al*., 2021; Chen *et al*., 2020; Liu *et al*., 2023; Monteagudo *et al*., 2017;

5 O’Donnell *et al*., 2015).

6 Information regarding many of these MLV vaccine candidates can be found in a recent review publication (Brake, 2022).

7 Different DIVA strategies using serological methods (e.g. ELISA) or genome detection methods (e.g. differential real-time

8 PCR) are not widely available for these ASF MLV first generation vaccine candidates. Therefore, there is still room for

9 improvement with respect to marker vaccines and their companion diagnostic tests.

10 Inactivated (non-replicating) whole virus vaccines are not presently available and may be difficult to develop to meet

11 minimum efficacy standards. Recombinant vectored, subunit vaccine candidates that can be produced in scalable vaccine

12 platform expression systems and mRNA-based ASF vaccines are being evaluated in ongoing laboratory research, testing

13 and evaluation in experimental challenge models*.*The publicly available *Center of Excellence for African Swine Fever*

14 *Genomics*(ASFV Genomics, 202243*)*that provides the structural protein predictions for all 193 ASFV proteins may help

15 accelerate ASF first and second generation vaccine research and development.

16 Fit-for-purpose vaccine use scenarios matched to the intended use in a domestic pig specific type of production system

17 may require different vaccine product profiles or may influence the focus of essential versus ideal vaccine requirements.

18 As with any MLV vaccine, all ASF MLV vaccines should be used according to the label instructions, under the strict control

19 of the country’s Regulatory Authority.

20 The minimum standards given here and in chapter 1.1.8 are intended to be general in nature and may be supplemented

21 by national, regional, and veterinary international medicinal product harmonised requirements. Minimum data requirements

22 for an authorisation in exceptional circumstances should be considered where applicable.

23 **2.	Outline of production and minimum requirements for vaccines**

##### 24 2.1.	Characteristics of the seed

###### 25 2.1.1.	Biological characteristics of the master seed

26 MLVs are produced from ASFV field strains derived from naturally attenuated field isolates or using

27 DNA homologous (genetically targeted) recombination techniques in cell cultures to delete one or

28 more ASFV genes or gene families. These molecular techniques typically involve replacement of the

29 targeted ASFV gene(s) with one or more positive, marker fluorescent (e.g. BFP, eGFP, mCherry) or

30 enzyme-based (e.g. β-glucuronidase) ASFV promoter-reporter gene systems that allow the use of

31 imaging microscopy or flow cytometry to visualise, select, and clone gene-deleted, recombinant, ASF

32 MLVs. MLV production is carried out in cell cultures based on a seed-lot system.

33 Master seed viruses (MSVs) for MLVs should be selected and produced based on their ease of

34 growth in cell culture, virus yield (log10 infectious titre) and genetic stability over multiple cell

35 passages. Preferably, a continuous well-characterised cell line (e.g. ZMAC-4; PIPEC; IPKM) (Borca

36 *et al*., 2021; Masujin *et al*., 2021; Portugal *et al*., 2020) is used to produce a master cell bank (MCB)

37 on which the MSV and MSV-derived working seed virus (WSV) can be produced. The exact source

38 of the underlying ASFV isolate, the whole genome sequence, and the passage history must be

39 recorded.

###### 40 2.1.2.	Quality criteria (sterility, purity, freedom from extraneous agents)

41 Only MSVs that have been established as sterile, pure (free of wild-type parental virus and free of

42 extraneous agents as described in Chapter 1.1.9 *Tests for sterility and freedom from contamination*

43 *of biological materials intended for veterinary use*, and those listed by the appropriate licensing

44 authorities) and immunogenic, should be used as the vaccine virus (WSV and vaccine batch

45 production). Live vaccines must be shown not to cause disease or other adverse effects in target

46 animals in accordance with chapter 1.1.8, Section 7.1 *Safety tests*(for live attenuated MSVs), that

47 includes target animal safety tests, increase in virulence tests, assessing the risk to the environment)

48 and if possible, no transmission to other animals.

49 Identity of the MSV must be confirmed using appropriate methods (e.g. through the use of vaccine

50 strain-specific whole genome detection methods such as next generation sequencing).

51 Demonstration of MSV stability over several cell passages is necessary, typically through at least five

52 passages (e.g. MSV+5). For those MLV vaccines for which attenuation is linked to specific

53 characteristics (gene deletion, gene mutations, etc.), genetic stability of attenuation throughout the

54 production process should be confirmed using suitable methods. Suitable techniques to demonstrate

55 genetic stability may include but are not limited to: genome sequencing, biochemical, proteomic,

56 genotypic (e.g. detection of genetic markers) and phenotypic strain characterisation. If final product

57 yields (infectious titres) are relatively low, genetic stability at a minimum of MSV+10 should be

58 demonstrated to allow more flexibility in the outline of production. For example, if MSV+8 is the

59 maximum passage for use in final product manufacturing, demonstration of genetic stability to at least

60 MSV+10 is warranted.

42 http://asfvgenomics.com. Accessed 4/4/2023.

###### 61 2.1.3.	Validation as a vaccine strain

62 The vaccine derived from the MSV must be shown to be satisfactory with respect to safety and

63 efficacy.

64 Even if pigs are not known for susceptibility to transmissible spongiform encephalopathy (TSE)

65 agents, consideration should also be given to minimising the risk of TSE transmission by ensuring

66 that animal origin materials from TSE-relevant species, if no alternatives exist for vaccine virus

67 propagation, comply with the measures on minimising the risk of transmission of TSE.

68 Ideally, the vaccine virus in the final product should generally not differ by more than five passages

69 from the master seed lot.

70 ASF vaccine should be presented in a suitable pharmaceutical form (e.g. lyophilisate or liquid form).

##### 71 2.2.	Method of manufacture

###### 72 2.2.1.	Procedure

73 The MLV virus is used to infect swine primary cell cultures obtained from specific-pathogen free pigs,

74 the requirements for which are defined in specific monographs (Chapter 2.3.3 *Minimum requirements*

75 *for the organisation and management of a vaccine manufacturing facility*, Section 2.4.2). Compared

76 with primary cell cultures, use of a continuous cell line generally allows for more consistency, higher

77 serial volumes in manufacturing and aligns better with a seed lot system. Thus, preferably a master

78 cell bank based established, continuous cell line shown to support genetically stable ASFV replication

79 and acceptable titres over several passages should be used.

80 Cell cultures shall comply with the requirements for cell cultures for production of veterinary vaccines

81 in chapter 1.1.8. Regardless of the production method, the substrate should be harvested under

82 aseptic conditions and may be subjected to appropriate methods to release cell-associated virus (e.g.

83 freeze–thaw cycles, detergent lysis). The harvest can be further processed by filtration and other

84 purification methods. A stabiliser or other excipients may be added as appropriate. The vaccine is

85 homogenised to ensure a uniform batch/serial.

###### 86 2.2.2.	Requirements for ingredients

87 All ingredients used for vaccine production should be in line with requirements in chapter 1.1.8.

###### 88 2.2.3.	In-process controls

89 In-process controls will depend on the protocol of production: they include virus titration of bulk

90 antigen and sterility tests.

###### 91 2.2.4.	Final product batch tests

92 i)  Sterility

93 Tests for sterility and freedom from contamination of biological materials intended for veterinary use

94 may be found in chapter 1.1.9.

95 ii) Identity

96 Appropriate methods such as specific genome detection methods (e.g. specific differential real-time

97 PCR) should be used for confirmation of the identity of the vaccine virus and differentiation from the parent strain of the virus as a potential contaminant.

**RATIONLE:**This phrase is added as explanatory to clarify the goal of the parenthetical (e.g., specific differential real-time PCR). Due to the genome size and methods of the vaccine virus generation, there is a biological (i.e., cell culture) phase present in every pathway of mutant generation in the lab. This leads to the potential for contamination with other viruses and the potential for contamination with parental (i.e., wild type, virulent) virus in the vaccine virus cultures. 

98 iii) Purity

99 Appropriate methods should be used to ensure that the final product batch does not contain any

100 residual wild-type ASFV.

101 iv) Safety

102 Batch safety testing is to be carried out unless consistent safety of the product is demonstrated and

103 approved in the registration dossier and the production process is approved for consistency in

104 accordance with the standard requirements referred to in chapter 1.1.8.

105 v) Batch/serial potency

106 Virus titration is a reliable indicator of vaccine potency once a relationship has been established

107 between the vaccine minimum immunising dose (MID) (minimum protective dose) and titre of the

108 modified live vaccine *in vitro*. In the absence of a demonstrated correlation between the virus titre

109 and protection, an efficacy test will be necessary (Section C.2.3.3 *Efficacy requirements*, below).

110 vi) Residual humidity/residual moisture

111 The test should be carried out consistent with VICH44 GL26 (*Biologicals: Testing of Residual*

112 *Moisture*, 200345). Required for MLV vaccines presented as lyophilisates for suspension for injection.

##### 113 2.3.	Requirements for authorisation/registration/licensing

###### 114 2.3.1.	Manufacturing process

115 For regulatory approval of a vaccine, all relevant details concerning history of the pre-MSV,preparation of MSV, manufacture of the vaccine and quality control testing (Sections C.2.1

116 *Characteristics of the seed*and C.2.2 *Method of manufacture*) should be submitted to the authorities.

117 Information shall be provided from three consecutive vaccine batches originating from the same MSV

118 and representative of routine production, with a volume not less than 1/10, and more preferably with

119 a volume not less than 1/3 of the typical industrial batch volume. The in-process controls are part of

120 the manufacturing process.

###### 121 2.3.2. Safety requirements

122 For the purpose of gaining regulatory approval, the following safety tests should be performed

123 satisfactorily.

124 As a minimum standard, vaccines should be tested for any pathogenic effects on healthy domestic

125 pigs of the target age intended for use. Additional demonstration of MLV safety in breeding age gilts

126 and pregnant sows is preferred but not required as a minimum standard.

127 i)	Safety in young animals

128 Carry out the test by each recommended route of administration using, in each case, piglets a

129 minimum of 6-weeks old and not older than 10-weeks old.

130 The test is conducted using no fewer than eight healthy piglets, and preferably no fewer than ten

131 healthy piglets.

132 Use vaccine virus at the least attenuated passage level that will be present in a batch of the vaccine.

133 Administer to each piglet a quantity of the vaccine virus equivalent to not less than ten times the

134 maximum virus titre (e.g. 50% haemadsorption dose [HAD50], 50% tissue culture infective dose

135 [TCID50], quantitative PCR, etc.) (maximum release dose) likely to be contained in one dose of the

136 vaccine. To obtain individual and group mean baseline temperatures, the body temperature of each

137 vaccinated piglet is measured on at least the 3 consecutive days preceding administration of the

138 vaccine.

139 To confirm the presence or absence of fever accompanied by acute and chronic disease, observe

140 the piglets 4 hours after vaccination and then at least once daily for at least 45 days, preferably 60

141 days post-vaccination. Carry out the daily observations for signs of acute and chronic disease using

142 a quantitative clinical scoring system adding the values for multiple clinical signs (e.g. Gallardo *et al.,*

143 2015a). These clinical signs should include fever, anorexia, recumbency, skin haemorrhage or

144 cyanosis, joint swelling and necrotic lesions around the joints, respiratory distress and digestive

145 findings).

146 At a minimum of 45 days post-vaccination, humanely euthanise all vaccinated piglets. Conduct gross

147 pathology on spleen, lung, tonsil, and kidney tissue samples and at least three different lymph nodes

43  VICH: International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medical Products

44 https://www.ema.europa.eu/en/documents/scientific-guideline/vich-gl26-biologicals-testing-residual-moisture-step-7_en.pdf

148 (which should include lymph node closest to site of inoculation, gastrohepatic and submandibular

149 nodes).

150 The vaccine complies with the test if:

151 	No piglet shows abnormal (local or systemic) reactions, reaches the pre-determined humane

152 endpoint defined in the clinical scoring system or dies from causes attributable to the vaccine;

153 	The average body temperature increase for all vaccinated piglets (group mean) for the

154 observation period does not exceed 1.5°C above baseline; and no individual piglet shows a

155 temperature rise above baseline greater than 2.5°C for a period exceeding 3 days.

156 	No vaccinated pigs show notable signs of disease by gross pathology

157 ii)	Safety test in pregnant sows and test for transplacental transmission

158 There is currently an absence of published information on ASFV pathogenesis in breeding-age gilts

159 and in pregnant sows associated with ASFV transplacental infection and fetus abortion/stillbirth. If a

160 label claim is pursued for use in breeding age gilts and sows, then a safety study in line with VICH

161 GL44 (*Guidelines on Target Animal Safety for Veterinary Live and Inactivated Vaccines*, *Section 2.2.*

162 *Reproductive Safety Test*, 2009 46) should be completed.

163 iii)	Horizontal transmission

164 The test is conducted using no fewer than 12 healthy piglets, a minimum of 6-weeks old and not older

165 than 10-weeks old and of the same origin, that do not have antibodies against ASFV, and blood

166 samples are negative on real-time PCR. All piglets are housed,, together from day 0 and the number

167 of vaccinated animals is the same as the number of naïve, contact animals. Co-mingle equal numbers

168 of vaccinated and naïve, contact piglets in the same pen or room.

169 Use vaccine virus at the least attenuated passage level that will be present between the master seed

170 lot and a batch of the vaccine. Administer by each recommended route of administration to no fewer

171 than six piglets a quantity of the vaccine virus equivalent to not less than the maximum virus titre

172 (maximum release dose) likely to be contained in 1 dose of the vaccine.

173 To obtain individual and group mean baseline temperatures, the body temperature of each naïve,

174 contact piglet is measured on at least the 3 consecutive days preceding co-mingling with vaccinated

175 piglets. The body temperature of each naïve, contact piglet is then measured daily for at least 45

176 days, preferably 60 days.

177 To confirm the presence or absence of fever accompanied by disease, observe the naïve, contact

178 piglets daily for at least 45 days, preferably 60 days. Carry out the daily observations for signs of

179 acute and chronic clinical disease using a quantitative clinical scoring system adding the values for

180 multiple clinical signs (e.g. Gallardo *et al.,*2015a). These clinical signs should include fever, anorexia,

181 recumbency, skin haemorrhage or cyanosis, joint swelling and necrotic lesions around the joints,

182 respiratory distress and digestive findings.

183 In addition, blood should be taken from the naïve contact piglets at least twice a week for the first 21

184 days post-vaccination and then on a weekly basis. From the blood samples, determine infectious

185 virus titres by quantitative virus isolation (HAD50/ml or TCID50/ml) and using a real-time PCR test. If

186 the vaccine virus is non-haemadsorbing or does not cause cytopathic effects, a real-time PCR test

187 only may be used.

188 Collect blood (serum) samples from the naïve contact pigs at least at day 21 and day 28 days and

189 carry out an appropriate test to detect vaccine virus antibodies. At a minimum of 45 days, humanely

190 euthanise all naïve, contact piglets. Conduct gross pathology on spleen, lung, tonsil, and kidney

191 tissue samples and at least three different lymph nodes. Determine virus titres in all collected samples

192 by quantitative virus isolation (HAD50/mg or TCID50/mg) and real-time(RT)-PCR (see Section B.1.

193 Identification of the agent). If the vaccine virus is non-haemadsorbing or does not cause cytopathic

194 effects, a real-time PCR test or other appropriate method (e.g. titration using IPT or FAT detection)

195 may be used.

45 https://www.ema.europa.eu/en/documents/scientific-guideline/vich-gl44-target-animal-safety-veterinary-live-inactived-vaccines-step-7_en.pdf.

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196 The vaccine complies with the test if:

197 	No vaccinated or naïve contact piglet shows abnormal (local or systemic) reactions, reaches the

198 predetermined humane endpoint defined in the clinical scoring system or dies from causes

199 attributable to the vaccine;

200 	The average body temperature increase for all naïve, contact piglets (group mean) for the

201 observation period does not exceed 1.5°C: above baseline; and no individual piglet shows a

202 temperature rise above baseline greater than 2.5°C for a period exceeding 3 days;

203 	No naïve, contact piglet shows notable signs of disease by gross pathology and no virus is

204 detected in their blood or tissue samples by virus isolation or real-time PCR.

205 

206 **RATIONALE:** It is important to be precise in acceptance criteria

207 	No naïve contact pigs test positive for antibodies to the vaccine virus.

208 iv)	Post-vaccination kinetics of viral replication (MLV blood and tissue dissemination) study

209 Prior to the reversion to virulence study (Section C2.3.2.v. below), a minimum of one study should be

210 performed to determine the post-vaccination kinetics of virus replication in the blood (viremia), tissues

211 and viral shedding.

212 The test consists of the administration of the vaccine virus from the master seed lot to no fewer than

213 eight healthy piglets, and preferably ten healthy piglets, a minimum of 6-weeks old and not older than

214 10-weeks old and of the same origin, that do not have antibodies against ASFV, and blood samples

215 are negative on real-time PCR.

216 Administer to each piglet, using the recommended route of administration most likely to result in

217 spread (such as the intramuscular route or intranasal route), a quantity of the master seed vaccine

218 virus equivalent to not less than the maximum virus titre (maximum release dose) likely to be

219 contained in 1 dose of the vaccine.

220 Record daily body temperatures and observe inoculated animals daily for clinical disease for at least

221 45 days, preferably 60 days.

222 Carry out the daily observations for signs of acute and chronic clinical disease using a quantitative

223 clinical scoring system adding the values for multiple clinical signs (e.g. Gallardo *et al.*(2015a). These

224 clinical signs should include fever, anorexia, recumbency, skin haemorrhage or cyanosis, joint

225 swelling and necrotic lesions around the joints, respiratory distress and digestive findings.

226 Collect blood samples from all the piglets at least two times per week from 3 days post-vaccination

227 for the first 2 weeks, then weekly for the duration of the test. Determine vaccine virus titres by

228 quantitative virus isolation (HAD50/ml or TCID50/ml) and using a real-time PCR test. If the vaccine

229 virus is non-haemadsorbing or does not cause cytopathic effects, a real-time PCR test only may be

230 used.

231 Determine which blood timepoint(s) should be used in the design of the reversion to virulence study

232 (Section C2.3.2.v. below).Collect oral, nasal and faecal swab samples (preferably devoid of blood to

233 minimise assay interference) at least two times per week from 3-days post-vaccination for the first

234 2 weeks, then weekly for the duration of the test. Test the swabs for the presence of vaccine virus.

235 Determine virus titres in all collected samples by quantitative virus isolation (HAD50/ml or TCID50/ml)

236 and using a real-time PCR test. If the vaccine virus is non-haemadsorbing or does not cause

237 cytopathic effects, a real-time PCR test or other appropriate method (e.g. titration using IPT or FAT

238 detection) may be used.

239 Euthanise at least two piglets on days 7, 14, 21, and preferably on day 28 (±2 days at each timepoint)

240 and collect spleen, lung, tonsil, kidney tissue samples and at least three different lymph nodes (which

241 should include lymph node closest to site of inoculation, gastrohepatic and submandibular nodes).

242 Determine virus titres in all collected samples by quantitative virus isolation (HAD50/mg or TCID50/mg)

243 and using real-time PCR test. If the vaccine virus is non-haemadsorbing or does not cause cytopathic

244 effects, a real-time PCR test or other appropriate method (e.g. titration using IPT or FAT detection)

245 may be used.

246 Determine which tissue(s) and timepoint(s) should be used to aid in the design of the reversion to

247 virulence study (Section C.2.3.2.v), for example, specific tissues at specific timepoints which show

248 the highest titres should be considered for selection and use in the reversion to virulence study.

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249 v)	Reversion to virulence

250 The test should be carried out consistent with VICH GL41 (Examination of live veterinary vaccines in

251 target animals for absence of reversion to virulence, 200847).

252 The test for increase in virulence consists of the administration of the vaccine master seed virus to

253 healthy piglets of an age (e.g. between 6-weeks and 10-weeks old) suitable for recovery of the strain

254 and of the same origin, that do not have antibodies against ASFV, and blood samples that are

255 negative on real-time PCR. This protocol is typically repeated five times.

256 *First pass*(*p1*)

257 Administer to no fewer than two piglets, and preferably no fewer than four piglets using the intended

258 route of administration for the final product, a quantity of the master seed vaccine virus equivalent to

259 not less than the maximum virus titre (maximum release dose) likely to be contained in 1 dose of the

260 vaccine. Observe inoculated animals daily for the appearance of at least two and preferably at least

261 three clinical signs and record daily body temperatures.

262 Based on results from at least one completed vaccine shed and spread (virus blood and tissue

263 dissemination study, Section C.2.3.2.iv above) collect an appropriate quantity of blood or tissue from each piglet on the predetermined single timepoint (day 5–13). Determine virus titres in individual blood samples by quantitative virus isolation (HAD50/ml or TCID50/ml) and by real-time PCR. If the vaccine virus is non-haemadsorbing or does not cause cytopathic effects, a real-time PCR test or other appropriate method (e.g. titration using IPT or FAT detection) may be used. Identify the individual sample(s) with the highest infectious titre and reserve for the subsequent *in-vivo*passage (second pass, p2).

264 Based on results from at least one completed vaccine virus blood and tissue distribution

265 dissemination study, Section C.2.3.2.iv above) euthanise piglets on the predetermined timepoint (i.e.

266 day 7, 14, 21, or 28). Determine infectious virus titres in individual blood and tissue samples by quantitative

267 virus isolation (HAD50/ml or TCID50/ml). If the vaccine virus is non-haemadsorbing or does not cause

268 cytopathic effects, a real-time PCR test or other appropriate method (e.g. titration using IPT or FAT

269 detection) may be used. Identify individual ~~tissue~~ sampletype(s) with the highest infectious titre. Pool the blood/tissues from different organs from all animals with the highest titres and prepare at least a 10% suspension in PBS, pH 7.2 kept at 4°C or at –70°C for longer storage. Test each ~~blood and tissue~~sample pool used for inoculation by PCR to confirm the absence of potential viral agent contaminants (i.e.,FMDV, PRRS, PCV2). Blood and pooled tissue (p1) are used to inoculate 2 ml of positive material using the intended route of administration for the final product to each of least two and ideally at least four further pigs of the same age and origin

**RATIONALE:** ASFV is recognized in several tissues in swine and is noted in this section above. Several edits provided as consistent statements that both blood and tissue are needed for studies. In fact, epithelial tissues, not blood, is not likely to cause the exposedisease transmission to other pigs. https://www.cfsph.iastate.edu/Factsheets/pdfs/african_swine_fever.pdf

270 *Second pass*(*p2*)

271 If no virus is found (p1), repeat the administration by the intended route once again with the same

272 pooled material (blood and pooled tissue, p1) in another ten healthy piglets of the same age and

273 origin.

274 If no virus is found at this point, end the process here. If, however, virus is found, carry out a second

275 series of passages by administering 2 ml of positive material using the intended route of

276 administration for the final product to each of no fewer than two piglets, and preferably no fewer than

277 four piglets of the same age and origin. Observe inoculated animals daily for the appearance of at

278 least two and preferably at least three clinical signs and record daily body temperatures.

279 *Third and fourth pass*(*p3 and p4*)

280 If no virus (p2), repeat the intramuscular administration once again with the same pooled material

281 (blood and pooled tissue, p2) in another eight healthy piglets of the same age and origin.

282 If no virus is found at this point, end the process here. If, however, virus is found, carry out this

283 passage operation no fewer than two additional times (p3 and p4) (to each of no fewer than two

284 piglets, and preferably no fewer than four piglets of the same age and origin) and verifying the

285 presence of the virus at each passage in blood and tissues. Observe inoculated animals daily for the

46 https://www.ema.europa.eu/en/documents/scientific-guideline/vich-gl41-target-animal-safety-examination-live-veterinary-vaccines-target-animals-absence-reversion_en.pdf.

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286 appearance of at least two and preferably at least three clinical signs and record daily body

287 temperatures.

288 *Fifth pass*(*p5*)

289 Administer 2 ml of the blood and pooled tissue (4) to each of at least eight healthy piglets of the same

290 age and origin. Observe inoculated animals daily for at least 28 days post-inoculation for the

291 appearance of at least two and preferably at least three clinical signs, and daily body temperature.

292 The vaccine virus complies with the test if:

293 	No piglet shows abnormal local or systemic reaction, reaches the pre-determined humane end

294 point defined in the clinical scoring system or dies from causes attributable to the vaccine; and

295 	There is no indication of increasing virulence (as monitored by daily body temperature

296 accompanied by clinical sign observations) of the maximally passaged virus compared with the

297 master seed virus.

298 At a minimum, a safe MLV vaccine shall demonstrate ALL the following features (minimal standards):

299 •	Absence of fever (defined as average body temperature increase for all vaccinated piglets (group

300 mean) for the observation period does not exceed 1.5°C above baseline; and no individual piglet

301 shows a temperature rise above baseline greater than 2.5°C for a period exceeding 3 days);

302 	Absence of chronic and acute clinical signs and gross pathology over the entire test period or

303 minimal chronic clinical signs (defined as mild swollen joints with a low clinical score that resolve

304 within 1 week).

305 •	Minimal (defined as no naïve, contact piglet shows notable signs of disease by clinical signs and

306 gross pathology and no or a low percentage of contact piglets test both real-time PCR positive

307 and seropositive) or no vaccine virus transmission (defined as no naïve, contact piglet shows

308 notable signs of disease by clinical signs and gross pathology and no contact piglets test both

309 real-time PCR positive and seropositive) over the entire test period;

310 •	Absence of an increase in virulence (genetic and phenotypic stability) (complies with the reversion

311 to virulence test).

312 In addition, the vaccines in their commercial presentation before being authorised for general use

313 should be tested for safety in the field (see chapter 1.1.8 Section 7.2.3). Additional field safety

314 evaluation studies may include but are not limited to: environmental persistence (e.g. determination

315 of virus recovery from bedding or other surfaces), assessment of immunosuppression, and negative

316 impacts on performance.

###### 317 2.3.3.	Efficacy requirements

318 i)	Protective dose

319 Vaccine efficacy is estimated in immunised animals directly, by evaluating their resistance to live

320 virus challenge. The test consists of a vaccination/challenge trial in piglets a minimum of 6-weeks old

321 and not more than 10-weeks old, free of antibodies to ASFV, and negative blood samples by real-

322 time PCR. The test is conducted using no fewer than 15 and preferably no fewer than 24 vaccinated

323 pigs, and no fewer than five non-vaccinated control piglets.

324 The test is conducted to determine the minimal immunising dose (MID) (also referred to as the

325 minimal protective dose [MPD] or protective fraction); using at least three groups of no fewer than

326 five and preferably not fewer than eight vaccinated piglets per group, and one additional group of no

327 fewer than five non-vaccinated piglets of the same age and origin as controls. Use vaccine containing

328 virus at the highest passage level that will be present in a batch of vaccine.

329 Each group of piglets, except the control group, is immunised with a different vaccine virus content

330 in the same vaccine volume. In at least one vaccinated group, piglets are immunised with a vaccine

331 dose containing not more than the minimum virus titre (minimum release dose) likely to be contained

332 in one dose of the vaccine as stated on the label.

333 Twenty-eight days (±2 days) after the single injection of vaccine (or if using two injections of the

334 vaccine then 28 days [±2 days] following the second injection), challenge all the piglets by the

335 intramuscular route. If previous studies have demonstrated acceptable efficacy using IM challenge,

Report of the Meeting of the Biological Standards Commission / September 2023

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336 then a different challenge route (e.g. direct contact, oral or oronasal) may be used. Challenged,

337 vaccinated piglets may be housed in one or more separate pens in the same room or in different

338 rooms. Challenged, naïve controls can be housed in one or more rooms that are separate from

339 challenged, vaccinated piglets.

340 Carry out the test using an ASFV representative strain of the epidemiologically relevant field strain(s)

341 where the vaccine is intended for use (e.g. ASFV B646L [p72] genotype II pandemic strain and other

342 p72 virulent genotype of recognised epidemiologic importance). For gene deleted, recombinant MLV

343 viruses, if neither challenge virus type is available, then carry out the test with the parental, virulent

344 virus used to generate the MLV recombinant virus. Use a 10e3–10e4 HAD50 (or TCID50 for non-HAD

345 viruses) challenge dose sufficient to cause death or meet the humane endpoint in 100% of the

346 nonvaccinated piglets in less than 21 days. Higher or lower challenge doses can be considered if

347 appropriately justified.

348 The rectal temperature of each vaccinated piglet is measured on at least the 3 days preceding

349 administration of the challenge virus, at the time of challenge, 4 hours after challenge, and then daily

350 for at least 28 days, preferably 35 days. Observe the piglets at least daily for at least 28 days,

351 preferably 35 days. Carry out the daily observations for signs of acute and chronic clinical disease

352 using a quantitative clinical scoring system adding the values for multiple clinical signs (e.g. Gallardo

353 *et al.*, 2015). These clinical signs should include fever, anorexia, recumbency, skin haemorrhage or

354 cyanosis, joint swelling and necrotic lesions around the joints, respiratory distress and digestive

355 findings.

356 Collect blood samples from the vaccinated challenged piglets at least two times per week from 3 days

357 post-challenge for at least 28, preferably 35 days. From the blood samples, determine infectious virus

358 titres by quantitative virus isolation (HAD50/ml or TCID50/ml) and using a real-time PCR test. If the

359 vaccine virus is non-haemadsorbing or does not cause cytopathic effects, a real-time PCR test only

360 may be used.

361 At the end of the test period, humanely euthanise all vaccinated challenged piglets. Conduct gross

362 pathology on spleen, lung, tonsil, and kidney tissue samples and at least three different lymph nodes.

363 (which should include lymph node closest to site of inoculation, gastrohepatic and submandibular

364 nodes). Determine virus titres in all collected samples by quantitative virus isolation (HAD50/mg or

365 TCID50/mg) and real-time PCR (see Section B.1. Identification of the agent). If the vaccine virus is

366 non-haemadsorbing or does not cause cytopathic effects, a real-time PCR test or other appropriate

367 method (e.g. titration using IPT or FAT detection) may be used.

368 The test is invalid if fewer than 100% of control piglets die or reach a humane endpoint.

369 The vaccine (or a specific vaccine virus dose if conducting a vaccine dose titration study) complies

370 with the test if:

371 	No vaccinated challenged piglet shows abnormal (local or systemic) reactions, reaches the

372 humane endpoint or dies from causes attributable to ASF;

373 	The average body temperature increase for all vaccinated challenged piglets (group mean) for

374 the observation period does not exceed 2.0°C above baseline; and no individual piglet shows a

375 temperature rise above baseline greater than 2.0°C;

376 	The vaccinated challenged piglets display a reduction or absence of typical acute clinical signs of

377 disease and gross pathology and a reduction or absence of challenge virus levels in blood and

378 tissues.

379 ii)	Assessment for horizontal transmission (challenge virus shed and spread study)

380 The ASF basic reproduction number, R0, can be defined as the average number of secondary ASF

381 disease cases caused by a single ASFV infectious pig during its entire infectious period in a fully

382 susceptible population (Hayes *et al*., 2021). In general, if the ASFV effective reproduction number

383 Re=R0 × (S/N) (S= susceptible pigs; N= total number of pigs in a given population) is greater than

384 1.0, disease is predicted to spread. Ideally, ASF vaccination should reduce Re to less than 1.0 by

385 reducing the number of susceptible, naïve, contact pigs exposed to vaccinated, infected pigs.

386 To evaluate ASF vaccine impact on ASF disease transmission, the test consists of a

387 vaccination/challenge trial in piglets a minimum of 6-weeks old and not older than 10-weeks old, free

388 of antibodies to ASFV, and negative blood samples by real-time PCR.

389 The test is conducted using no fewer than 15 healthy piglets at a ratio comprising twice the number

390 of vaccinated piglets to naïve piglets (e.g. ten vaccinated and five naïve). Use vaccine containing

391 virus at the highest passage level that will be present in a batch of the vaccine.

392 The quantity of vaccine virus administered to each pig is equivalent to be not more than the minimum

393 virus titre (minimum dose) likely to be contained in one dose of the vaccine as stated on the label.

394 Following immunisation, vaccinated and naïve piglets should continue to be co-mingled.

395 Twenty-eight days [±2 days] after the single injection of vaccine (or if using two injections of the

396 vaccine then 28 days [± 2 days] following the second injection), temporarily separate [into different

397 pen(s) or room(s)] all vaccinated piglets from naïve piglets. Challenge all vaccinated piglets by the

398 intramuscular or other previously verified route. Carry out the challenge using an ASFV

399 representative strain of the epidemiologically relevant field strain(s) where the vaccine is intended for

400 use (e.g. ASFV B646L [p72] genotype II pandemic strain and other p72 virulent genotype of

401 recognised epidemiological importance). For gene deleted, recombinant MLV viruses, if neither

402 challenge virus type is available, then carry out the test with the parental, virulent virus used to

403 generate the MLV recombinant virus. Use a 10e3–10e4 HAD50 (or TCID50 for non-HAD viruses

404 challenge dose sufficient to cause death or met the humane endpoint in 100% of the nonvaccinated

405 piglets in less than 21 days. Higher or lower challenge doses can be considered if appropriately

406 justified.

407 Approximately 18-24 hours later, re-introduce naïve piglets to vaccinated, challenged piglets and

408 allow for direct nose to nose contact exposure with vaccinated, challenged piglets. Allow for

409 continuous contact exposure by co-mingling both groups through the end of the study. If more than

410 one pen or room is used for co-housing, following reintroduction initially maintain a ratio of 2:1 of

411 challenged, vaccinated piglets to contact exposed, naïve piglets.

412 The rectal temperature of each contact piglet is measured on at least the 3 days preceding

413 administration of the challenge virus to vaccinated pigs, immediately prior to direct contact exposure,

414 4 hours post-contact exposure, and then daily for at least 28, preferably 35 days. Observe all contact

415 exposed piglets at least daily for at least 28 days, and preferably for at least 35 days.

416 Carry out the daily observations in each contact piglet for signs of acute and chronic clinical disease

417 using a quantitative clinical scoring system adding the values for multiple clinical signs (e.g. Gallardo

418 *et al.,*2015a). These clinical signs should include fever, anorexia, recumbency, skin haemorrhage or

419 cyanosis, joint swellingand necrotic lesions around the joints, respiratory distress and digestive

420 findings.

421 In addition, blood should be taken from the naïve contact piglets at least twice a week from 3 days

422 post-contact exposure for the duration of the test period. From the blood samples, determine

423 infectious challenge virus titres by quantitative virus isolation (HAD50/ml or TCID50/ml) and using a

424 real-time PCR test. If the vaccine virus is non-haemadsorbing or does not cause cytopathic effects,

425 a real-time PCR test only may be used.

426 Collect blood (serum) samples from the naïve contact pigs at least at day 21 and day 28 (±2 days),

427 and at the end of the test period, and carry out an appropriate test to detect vaccine virus antibodies.

428 Collect oral, nasal and faecal swab samples (preferably devoid of blood to minimise assay

429 interference) from all contact-exposed naïve piglets at least two times per week from 3-days post-

430 contact exposure for the first 2 weeks, then weekly for the duration of the test and test swabs for the

431 presence of challenge virus. Determine virus titres in all collected samples by quantitative virus

432 isolation (HAD50/ml or TCID50/ml) and using a real-time PCR test. If the vaccine virus is non-

433 haemadsorbing or does not cause cytopathic effects, a real-time PCR test or other appropriate

434 method (e.g. titration using IPT or FAT detection) may be used.

435 At the end of the test period, humanely euthanise all contact piglets. Conduct gross pathology on

436 spleen, lung, tonsil, and kidney tissue samples and at least three different lymph nodes. (which should

437 include lymph node closest to site of inoculation, gastrohepatic and submandibular nodes).

438 Determine virus titres in all collected samples by quantitative virus isolation (HAD50/mg or TCID50/mg)

439 and real-time PCR (see Section B.1. Identification of the agent). If the vaccine virus is non-

440 haemadsorbing or does not cause cytopathic effects, a real-time PCR test or other appropriate

441 method (e.g. titration using IPT or FAT detection) may be used.

**RATIONALE:** Period of validity not defined anywhere in this document. 

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709 eu.net/en/Home/Downloads.htm

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813 10.1128/JVI.01199-21.

814	*

815	* *

816	**NB:**There are WOAH Reference Laboratories for African swine fever

817	(please consult the WOAH Web site:

818	https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).

819	Please contact the WOAH Reference Laboratories for any further information on

820	diagnostic tests and reagents for African swine fever

821

822

**NB:**FIRST ADOPTED IN 1990. MOST RECENT UPDATES ADOPTED IN 2021.

823

824

825

826

827

828

**Appendix 1 to Annex 16 of the Report of the Biological Standards Commission meeting 4–8 September 2023**

**Draft Standards for African Swine Fever (ASF) Modified Live Virus (MLV) Vaccines for Domestic and Wild Pigs**

Annex16_3.09.01_ap pendix1_GL_ASF_MLV

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