Advanced Biotechnology in the Development of Novel Vaccines against Major Farm Animal Infectious Diseases in China

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Diseases in China Xiufan Liu Animal Infectious

1 Advanced Biotechnology in the Development of Novel Vaccines against Major Farm Animal Infectious Diseases in China Xiufan Liu Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University

2 China has experienced a flourishing period in farm animal industries since 1980, and the annual output of the major farm animal products including meats, poultry eggs, milk and wool has increased rapidly in the past 30 years.

3 Yearly production of major farm animal products between in China (million tons) Year Meat Poultry eggs Milk Wool Fold increase* Data source: China statistics year book 2013; China animal husbandry statistics 2013 Fold increase*: 2012/1980

4 Increase of the per capita annual consumption of meat, poultry eggs and milk (kg) in China between Notes :average population size

5 Emerging and reemerging devastating infectious diseases in farm animals in China during Disease Year of first outbreak Affected spices ref H9N2 Avian influenza 1994 Poultry (chicken, quail, waterfowl) H5N1 HPAI 1996 Poultry, wild birds, some mammals Chen et al Xu et al Genotype VIId ND 1997 Poultry, wild birds Rui et al Very virulent infectious basal disease (IBD) 1998 Poultry Cui et al H7N9 avian influenza 2013 Sporadic cases in humans, poultry infection O serotype, Pan Asia Subtype foot-and mouth disease (FMD) Serotype Asian l foot-and mouth disease (FMD) 1999 Cattle, swine sheep goat, deer (cloven-hoofed) Gao et al Cloven-hoofed animals Serotype A foot-and mouth disease (FMD) 2009 Cloven-hoofed animals Porcine reproductive and respiratory syndrome (PRRS) 1995 Swine Yang et al PCV2 infections 2000 Swine Lang et al Highly pathogenic PRRS 2006 Swine Tian et al Porcine epizootic diarrhea (PED) 2010 Swine Yu et al New subtype pseudorabies 2012 Swine Tong et al Peste des petits ruminants (PPR) 2007 Sheep, goat However, the rapid development and growth in farm animal industries bring about several challenges as well, one of which is animal disease problem we are confronting.

6 Vaccines are useful tool in fighting against infectious diseases and vaccination is one of the most cost-effective strategies in the prevention, control and eradication program.

Disadvantages of conventional live vaccines 1. Residual virulence; 2. Reversion of vaccine strains to pathogenic wild type; 3. Source of environmental contamination; 4.

7 Disadvantages of conventional live vaccines 1. Residual virulence; 2. Reversion of vaccine strains to pathogenic wild type; 3. Source of environmental contamination; 4. Unable to cope with the prevailing field strains; 5. Not suitable for pathogens that infect immune cells; 6. Not DIVA (Differentiate infected from vaccinated animals)

8 Disadvantages of conventional killed vaccines 1. No CTL response induced, less protective; 2. Require adjuvants and several injections; 3. Effective only in controlling clinical signs rather than infection; 4. Not suitable for pathogens that cannot grow well in cell culture; 5. More expensive; 6. Always with side effect, mild or severe; 7. Not DIVA (Differentiate infected from vaccinated animals)

9 Disadvantages Because of the disadvantages and limitations of the conventional live and killed vaccines as well as for the control of emerging and re-emerging pathogens, new vaccines are greatly demanded

10 Licensed new vaccines by advanced biotechnology for farm animal use in China Vaccine Species Disease Ref. Gene-deleted PRV Swine Pseudorabies Chen et al rfpv+aiv/h5-ha-na Chicken FP, HPAI Qiao et al rfpv+iltv/gb Chicken FP, ILT Wang et al rndv+aiv/h5-ha Chicken ND, HPAI Chen et al RGS-Modified H5N1 vaccine Chicken HPAI Chen et al IBDV VP2 subunit Chicken IBD Du et al Synthesized FMDV peptide (O) Swine FMD Sun et al Chlamydia psittaci, subunit Chicken Psittacosis Liu et al LT/LTB (E. coli) subunit Cattle, sheep Diarrhea Wang et al In this review we will summarize the novel vaccines which have been licensed and in development by advanced biotechnology in the past 15 years in China. These vaccines target important pathogens of farm animals, including viruses, bacteria and parasites.

11 New vaccines by advanced biotechnology for farm animal use in the process of licensing in China Vaccine Species Disease Ref. PCV2 subunit Cap (E. coli) Swine PCV2 infection Fan et al H5 AIV HA DNA (clade 0) chicken, waterfowl H5 HPAI Jiang et al Genotype VII ND (RGS) chicken, goose ND Hu et al APP gene deleted (apxiic-/apxic) - Swine Pleuropneumonia Bei et al Goatpox virus vectored goat, sheep peste des petits Wang et al PPRV ruminants Dual marker PRRS vaccine swine highly pathogenic PRRS Xu et al (RGS-based) Modified FMDV type A vaccine cloven-hoofed Type A FMD Zheng et al

12 New vaccines by advanced biotechnology for farm animal use in development in China Vaccine Species Disease ref. Chimera PCV1-PCV2 Cap Swine PCV2 infection Gao et al Chimera CSF-JE viral replicon Swine CSF, JE Yang et al MDV Meg oncogene deleted Chicken MD Su et al MDV vectored H9N2 vaccine Chicken MD, H9N2 AI Zhang et al FMD VLP of VP0, VP1, VP3 cloven-hoofed FMD Asia I Guo et al SPV-PCV2 Capsid Swine PCV2 Lin et al spic gene-deleted Salmonella Chicken pullorum Disease Geng et al DEV vectored H5 AI vaccine poultry H5 AI Liu et al Chimeric FMDV A in O cloven-hoofed FMD type A Zheng et al

13 The Classification of the novel vaccines Genetically modified vaccines Live viral and bacterial vector vaccines Subunit /recombinant protein vaccines Virus-like particle (VLP) vaccines According to their different features and specific techniques used DNA vaccines

14 Genetically modified vaccines Gene deleted viral vaccines MDV Meq gene deleted vaccine Gene deleted bacterial vaccines Salmonella gallinarum spic gene-deleted vaccine Viral chimera vaccines; Chimeric CSF and JE VRP vaccine Reverse genetics based (RGB) gene modified vaccines Genotype VII NDV vaccine

15 Genotype VII NDV vaccine: better match to the prevailing field strains in China Genotype VIId viruses are genetically and antigenically remote from the most extensively used vaccine strain LaSota that belongs to genotype II NDV. Gene F HN ND is endemic in China while the predominant circulating NDVs are genotype VIId viruses Identities 84.1%~84.4% 78.7%~83.0%

16 Generation of genotype VIId NDV vaccine by reverse genetics Liu, et al. Arch Virol, 2007 Hu, et al.vaccine, 2009

17 Biological characteristics of genotype VIId vaccine strain Viruses Pathogenicity Allantoic fluids titer MDT ICPI EID 50 /0.1ml HA E1 >120h log2 E5 >120h log2 E10 >120h log2 E15 >120h log2 Genotype VIId vaccine strains (A-VII) exhibits lower MDT and ICPI values than LaSota, and has high virus titer in embryonated eggs.

18 HI titers at different times post vaccination * HI titers were determined using homologous antigens. Inactivated A-VII vaccine can induce high antibody titers at a low dose (20μl) in SPF chickens and HI titers rise faster than that elicited by LaSota.

19 Immune efficacy of NDV/A-VII in chickens Frequency of isolation of the challenged virus in commercial chickens Post-challenge samples (no. positive/total) Group a Day 2 Day 4 Day 7 Day 10 O b C c O C O C O C PBS-C 10/10 10/10 10/10 10/10 NS d NS NS NS L-A-VII-C 8/10 3/10 1/10 3/10 0/10 1/10 0/10 0/10 L-Las-C 10/10 3/10 3/10 6/10 0/10 2/10 0/10 0/10 Oil-A-VII-C 5/10 * 0/10 2/10 2/10 0/10 1/10 0/10 0/10 Oil-Las-C 9/10 0/10 4/10 4/10 1/10 0/10 0/10 0/10 a C: challenged with 10 5 ELD50 JS2/06. b O: Oropharyngeal swabs. c C: Cloacal swabs. d NS: no survivors.

20 (A) Mean virus titers of oropharyngeal swabs of immunized groups on all sampling days. (B) Comparison of oropharyngeal virus titers at 2 day post-challenge. (A) Mean virus titers of cloacal swabs of immunized groups on all sampling days. (B) Comparison of cloacal virus titers at 4 day post-challenge. Hu, et al.vaccine, 2009

Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development The pol I/pol II unidirectional transcription system to make infectious FMDV clone by RGS.

21 Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development The pol I/pol II unidirectional transcription system to make infectious FMDV clone by RGS. Making chimeric FMD (ra/fmdv) by replacing ro/cha/99 P1 gene with P1 from field virus A/WH/CHA. ra/fmdv not only grows well the same as ro/cha/99 in BHK cells, but has excellent antigenic matching against serotype A FMD Zheng et al, PLOS One, 2012

22 Live viral vector vaccines: 1. Adenovirus vectors; 2. Poxvirus vectors; Fowlpox virus (FPV), Canarypox virus, Swinepox virus (SPV) 3. Herpesvirus vector Bovine herpesvirus (BHV), Marek s disease virus (MDV), HVT, Duck enteritis virus (DEV) Pseudorabies virus (PRV), 4. RGB viral vectors

23 DEV genome structure and 5 fosmid DNA fragments Construction of the fosmid with HA inserted within the ul41 gene Construction of the fosmid with HA inserted between the us7 and us8 genes. Recombinant H5 HPAI vaccine with DVE as the vector, Liu et al, J Virol, 2011

24 Efficacy of DEV-H5 HPAI recombinant live vaccine

25 Live bacterial vector vaccines 1. Salmonella spp. Vectors 2. Bacillus subtilis vector: FMDV multi-epitopes 3. Lactobacillus casae and Lactococcus lactis vectors IBV, PPV, PRRSV, PEDV 4. other bacteria vectors

26 Subunit/recombinant vaccines: 1. Recombinant proteins produced in various expression systems: E. coli; Baculovirus-insect cell; Pichia pastoris 2. Virus-like particle vaccines very safe (without nucleic acid); highly immunogenic; can induce CTL response

27 Virus-like particle (VLP) vaccines Main stages of self-assembly of VLPs using the BES. Liu et al (a) Both Linearized AcNPV baculovirus DNA and recombinant transfer vector are co-transfected into Sf cell and recombination occurs. (b) Recombinant baculovirus is produced within the cell. (c) Recombinant viruses are harvested and amplified to infect insect cells. (d) The foreign genes express the proteins of interest, respectively. (e) The proteins of interest self-assemble into VLPs by interaction with each other within the cytoplasm. (I) recombinant transfer vector (II) linearized AcNPV baculovirus DNA (III) recombinant baculovirus DNA (IV) recombinant baculovirus (V) proteins of interest.

28 Schematic diagram of separate pathways for taking, processing and presentation of enveloped VLP-derived antigens. A VLP is taken up by the antigen presenting cell (APC) via either endocytosis (route I) or receptor-mediated fusion (route II): the ternalized VLP (route I) is processed and presented via the class II MHC pathway; the other VLP (route II) is taken up by the APC, and subsequently presented via the class I MHC pathway. Therefore, the VLP vaccines have the advantage to strongly induce both humoral and CTL responses. Liu et al. 2012

, Virus Res 2013 FMDV-specific immune responses in pigs: A.

29 FMDV VLP containing VP0, VP3 and VP1 produced by SUMO fusion protein system in E. coli Guo et al., Virus Res 2013 FMDV-specific immune responses in pigs: A. Virus specific Ab titer; B. neutralizing Ab titer; C. gamma IFN production by PMBC

30 DNA vaccines: 1. Conventional plasmid DNA vaccines; 2. Alphavirus replicon-based suicidal DNA vaccines 3. DNA vaccines delivered by attenuated No reversion of virulence; long lasting immunity, both humoral and cellmedicated; less environment problem in production. Salmonella or other vehicles

31 Generation of a codon optimized DNA vaccine for H5N1 HPAI Primer About 200nt 20nt nt Oligo Overlapping PCR 20nt Primer About 200nt Bluescript M13-F1 ligation Bluescript M13-F2 Bluescript M13-HA Sequence

32 Antibody response of SPF chickens immunized with pcaggoptiha5 HI titers(log2) µg DNA, two doses, 3 weeks interval 1 0 w1 w2 w4 w6 w8 w10 w12 w14 w16 w18 w20 w22 w24 w26 w28 w30 w32 w34 w36 w38 w40 w42 w44 w46 w48 W50 w52 weeks after immunization Immunized Control Jiang et al, Antiviral Res, 2007

33 10 9 Antibody response of commercial chickens immunized with pcaggoptiha5 8 HI titers(log2) Boost Boost w1 w2 w4 w6 w8 w10 w12 w14 w16 w18 w20 w22 w24 w26 w28 w30 w32 weeks after immunization w34 w36 w38 w40 w42 w44 w46 w48 w50 w52

34 Codon optimized pcaggoptiha vaccine Homologous challenge Heterologous challenge Jiang et al, Antiviral Res, 2007

35 DIVA vaccines DIVA or marker vaccines, any one of the above novel vaccine can be DIVA vaccine when companion diagnostic kit is applied. To implement a disease control and eradication program, a DIVA vaccine is a must!

36 Dual marker vaccine against highly pathogenic porcine reproductive and respiratory syndrome Nsp2 1a 1b From NDV Xu and Tong et al, Vet Microbiol, 2012

The positive marker is used to identify the vaccinated animals, and the negative marker is used to identify the animals infected

37 ELISA to detect Abs to PRRSV Detection of Ab to positive marker NDV NP49 gene in the immunofluorescence assay A marker vaccine should contain two different markers: positive marker and negative marker. The positive marker is used to identify the vaccinated animals, and the negative marker is used to identify the animals infected with wild type virus. Xu and Tong et al, Vet Microbiol, 2012 ELISA to detect Abs to positive marker NDV NP49 ELISA to detect Abs to negitive marker: the deleted 25 aa in nsp2 of PRRSV

38 China has implemented the National Medium-term and Long-term Prevention and Control Program of Animal Infectious Diseases ( ) since 2012 and comprehensive strategies including enforced biosecurity, culling of infected animals, restriction of animal and animal product transportation, quarantine, disinfection and vaccination are in place. It is certain that the development of novel vaccines will speed up the advancement of this program.

39 Thank you!

The humoral immune responses to IBV proteins.

The humoral immune responses to IBV proteins. E. Dan Heller and Rosa Meir The Hebrew University of Jerusalem, Israel COST FA1207 meeting WG2 + WG3, Budapest, Jan. 2015 1 IBV encodes four major structural