Implication of antigenic variation for vaccine development: the Anaplasma marginale model. Kelly A. Brayton

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1 Implication of antigenic variation for vaccine development: the Anaplasma marginale model Kelly A. Brayton

2 Order Rickettsiales Family Anaplasmataceae Rickettsiaceae

3 Anaplasma marginale Causes anaplasmosis, an important disease of cattle with no available vaccine Infects the red blood cell Anemia, weight loss, abortion, death Obligate intracellular organism

4 Persistent infection cyclic bacteremia Organisms/ml blood acute persistent Kieser et al., II 1990

5 MSP2 surface coat variants emerge during sequential bacteremic cycles MSP2 Organisms/ml blood emergence control P1 P2 P3 French et al., II 1998, 1999 acute persistent

6 MSP2 opag3 opag2 opag1 msp2 Immunodominant surface protein ~36 kd Central HVR Operon linked expression Multigene family Barbet et al., II 2000 opag1 opag2 opag3

7 All other copies of msp2 are functional pseudogenes opag3 opag2 opag1 msp2 Sequenced St Maries strain 1 ES 7 functional ψ Brayton et al., PNAS 2001, 2005

8 Analysis of ES variants Sampled weekly though acute infection Ticks transmit only 2 MSP2 variants of the South Idaho strain

9 Segmental Gene Conversion SGV2 GGARVEVEVGYERFVIKGGKKSNEDTASVFLLGKELAYDTARGQVDRLATALGKMTKGEAKKWGNAIESATG Ψ A3 GGARVEVEVGYERFVIKGGKKSNEDTASVFLLGKELAYDTARGQVDRLATALGKMTKSEAKKWGNAIESVTG A3-1 GGARVEVEVGYERFVIKGGKKSNEDTASVFLLGKELAYDTARGQVDRLTNALGKMTKSEAKKWGTTVEAAT- A3-2 GGARVEVEVGYERFVIKGGKKSNEDTASVFLLGKELAYDTARGQVDRLTNALGKMTKSEAKKWGTTVEAAT- A3-3 GGARVEVEVGYERFVIKGGKKSNEDTASVFLLGKELAYDTARGQVDRLTNALGKMTKSEAKKWGTTVEAAT- BLOCK 1 SGV2 TTNGEKVSQKVCGNGTGSSG TTQRKISEVFTSDTETAQLSTMENTSTTSGATISTSGMAGN Ψ A3 TTNGQTVSQKVCGKGEGSNGTKKCGTNDGTTATQHKISEVFTEGTDTATL------LSAAGDTINTTGMAGN A3-1 --NGQTVSQKVCGKGEGSNGTKKCGTNDGTTATQHKISEVFTEGTDTATL------LSAAGDTINTTGMAGN A3-2 --NGQTVSQKVCGNGTGSNCGVNSGTTG--STTQHKISEVFTEGTDTATL------LSAAGDTINTTGMAGN A3-3 --NGQTVSQKVCGNGTGSNCGVNSGTTG--STNGNKISAVFSAEGAEAISS---MDTTSNGTTINVSGMATN BLOCK 2 BLOCK 3 Short sequential changes are made Brayton et al., Mol Micro 2002

10 Segmental gene conversion generates complex variants Expression site pseudogenes Ψ1 X Ψ2 ΨP1 simple Ψ9H1 ΨE6/F7 complex

11 Increasing complexity over time 3 Mean number of Segmental changes Mean number of segmental changes Months after tick transmission Months after tick transmission 0: whole ψ 2: two segments from different ψ 1: single segment 3: three segments from different ψ

12 Anchoring model X X X X X X X X Futse et al., Mol Micro 2005

13 Anchoring model Generates many more variants than a sequence identity model 4 5 sequence combinations Limits the requirement for sequence identity to the conserved 5 and 3 domains Predicts that strains would diverge in msp2 pseudogene and thus variant repertoire

14 Paradox: Persistently infected animals cannot clear the existing pathogen--yet are immune to infection with a second genotype (strain superinfection)

15 The basis for the paradox lies in the mechanism of msp2 variation Infected animals generate immune responses against the MSP2 variants initially variants represented by whole ψ and single segmental changes With time, immunity against a broad repertoire of variants develops and is maintained During persistent infection, complex mosaics, not represented in any single ψ, are generated and allow continued evasion of immune clearance New infections (strain superinfection) are cleared as the memory response recognizes the expressed MSP2 variants Does not allow the multiple recombination events needed to generate a unique mosaic capable of evasion

16 The basis for the paradox lies in the mechanism of msp2 variation SIMPLE COMPLEX

17 7285 ; 6DE ; 6DE ; 6DE ; 4B ; 5B DE DE DE DE DE D B B B B B B B B 3261 msp1α genotype PCR ELISA Animal msp1α genotype PCR ELISA Animal Dual infection (6%) Palmer et al., J Clin Micro, 2004

18 EMΦ 5B 5B 6B 6DE St. M Assignment of sequence to each msp2ψlocus G G E6/F7 E6/F7 E6/F7 E6/F7 E6/F G ES ES ES ES ES ES Rodriguez et al., Gene H1

19 EMΦ 5B 5B 6B 6DE St. M Association between dual infection and msp2 repertoire G G E6/F7 E6/F7 E6/F7 E6/F7 E6/F G ES ES ES ES ES ES Rodriguez et al., Gene H1

20 Hypothesis: Superinfection will occur among strains with diverse msp2 pseudogenes

21 EMΦ StM strain Challenge experiment Ho: strains with completely different msp2 pseudogene repertoires will superinfect 46 EMΦ 8 St. M G year E6/F7 G11 1 ES ES St Maries strain EMΦ strain 4 9H1

22 Superinfection results Track superinfection status with specific PCR for msp1α genotype.

23 6DE StM strain Challenge 2 How many different pseudogenes are required for superinfection? 6DE G11 2 G11 2 St. M year E6/F7 E6/F7 G ES ES StM strain 6DE strain 42 9H1

24 Results = Superinfection 6DE St. M St. M 6DE Usage of 6DE specific ψ 9H1 ψ usage

25 Summary Strains with as few as 1 novel pseudogene can superinfect providing the novel pseudogene is used in the ES Establishes the importance of the msp2 allelic repertoire in initial infection Would predict the evolution of an allelic repertoire as diverse as possible

26 Hypothesis: There is selection for growth fitness independent of immune evasion St. Maries PI Naïve calf Direct inoculation Measurement of variant complexity Input (from persistent infection): 2.1 Day 7 post-infection: 0.29 Day 12 post-infection: 0.62 Palmer et al., I&I 2007

27 Msp2 ψ repertoire Under competing evolutionary forces Selection for growth fitness Selection for immune evasion

28 How diverse is msp2?

29 Implications for vaccine strategy Simple variants predominate in early infection Immunize against simple variants protection? Subdominant epitopes

30 Acknowledgements Guy H. Palmer Donald P. Knowles Glen Scoles Bev Hunter Ralph Horn Xiaoya Cheng James E. Futse Christina K. Leverich Jose-Luis Rodriguez Michael J. Dark Pei-Shin Ku

31 Funding NIH NIAID RO1 AI45580 & AI44005 Wellcome Trust GR075800M USDA CSREES

TRANSMISSION DYNAMICS OF GENETICALLY DISTINCT STRAINS OF. Anaplasma marginale FOLLOWING SUPERINFECTION MARIA FERNANDA BANDEIRA DE MELO GALLETTI

TRANSMISSION DYNAMICS OF GENETICALLY DISTINCT STRAINS OF Anaplasma marginale FOLLOWING SUPERINFECTION By MARIA FERNANDA BANDEIRA DE MELO GALLETTI A thesis submitted in partial fulfillment of the requirements