Identification of vaccine candidate antigens for a Babesia bovis transmission blocking vaccine

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Suarez Animal Disease Research Unit ARS-USDA Department of

1 Identification of vaccine candidate antigens for a Babesia bovis transmission blocking vaccine Carlos E. Suarez Animal Disease Research Unit ARS-USDA Department of Veterinary Microbiology and Pathology Washington State University IDRC-Canada

bigemina more significant in tropical and semitropical

2 Bovine babesiosis Bovine babesiosis agents: B. bovis B. bigemina B. divergens. B. bovis and B. bigemina more significant in tropical and semitropical regions worldwide Mainly transmitted by Rhiphicephalus microplus and R. annulatus

3 Bovine babesiosis Babesia: intra-erythocytic apicomplexan parasites responsible of acute and persistence disease B. bovis more virulent that B. bigemina Acute signs: severe anemia, fever, anorexia, ataxia, respiratory distress syndrome, neurological signs, etc.

4 Control of B. bovis -Babesicides (ie: imidocarb) -Control of tick populations -Vaccination using live attenuated parasites -Acaricides -Pasture vacation -Vaccine based on tick antigen New vaccines are needed No transmission blocking (TB) vaccine available

5 Author: Alan R Walker

6 Sexual forms and gametocytes express a differential set of surface expose antigens: TARGETS

7 Toolbox development and the identification of transmission factor targets towards developing Babesia bovis non-transmissible vaccine strains and subunit TB vaccines

8 ID of transmission factors targets in B. bovis Initial focus on 6-Cys gene family and HAP-2, proteins Tools: 1] Transfected parasite lines lacking key 6-cys and HAP-2; 2] in vitro model to study sexual stage transitions and preliminary evaluations of the vaccine candidates.

9 The 6-cys family of B. bovis

10 The 10-member 6-cys family of B. bovis

11 Differential expression of the B. bovis 6-Cys gene family Blood stages Tick stages The 6-cys genes A, B are markers of B. bovis tick stages

12 Expression of 6-cys A and B in B. bovis stages present in the midgut of infected ticks Antibodies against the 6-cysA and 6-Cys B proteins recognize a ~60 kda antigen in midguts of female adult ticks feeding on cattle experimentally infected with B. bovis

13 6-Cys A and B are potential subunit TB-vaccine candidates Single-copy genes Limited polymorphism and high conservation among strains lack of expression in the bovine host Expression during tick stages Signal peptides Redundant function [Seq. similarities, domain distribution and location]

14 The HAP 2 gene Conserved among many organisms: in Plasmodium HAPLESS2/GCS1 (HAP2) is exclusively expressed on the surface of microgametes. HAP2 is critical for the fertilization of malaria parasites prior to development of the stage that infects mosquito gut epithelial cells. In Plasmodium, HAP2 is a candidate for a transmission blocking vaccine. The multi-intron, single copy, B. bovis hap2 gene encodes for a ~84 kda cell membrane protein with a signal peptide (fusogenic protein?)

15 The B. bovis HAP-2 gene is also differentially expressed in tick midgut forms

16 Transfection as a Babesia research tool

17 Some possible applications of transfection approaches for developing TB-Babesia vaccines 1. Functional analysis of candidate genes 1. Production of transfected non-transmissible KO-vaccine strains. 2. Expression of strain markers that tag attenuated parasites (live vaccine strain markers. 3. Expression of vaccine-relevant genes, such as tick protective antigens or stage-specific antigens of Babesia, such as sexual stage or kinete stage proteins in blood stage transfected parasites:

Phenotype Genotype PPE 6Cys E Wild type T3Bo strain 6Cys E E-KO clone 12 10 8 6 4 2 0-2 Comparative in

18 Phenotype Genotype PPE 6Cys E Wild type T3Bo strain 6Cys E E-KO clone Comparative in vitro WT T3Bo strain Days in blood culture Alzan et al., Parasit Vectors May 2;10(1):214

19 3878 bp A] Intergenic region Bbo E 6-Cys gene Intergenic region 5 flanking 3 flanking B] Ef-1 -B 5 flanking egfp bsd 3 rap-1 3 flank ing promoter p6-cys-eko C] Intergenic region Ef-1 -B 5 flanking egfp bsd 3 rap-1 3 flank ing promoter Intergenic region 7117 bp Alzan et al., Parasit Vectors May 2;10(1):214

20 Expression of GFP in B. bovis E-KO parasites A] B] Alzan et al., Parasit Vectors May 2;10(1):214

21 Parasitemia % Chromosome # Functional analysis of 6-Cys genes using double-ko transfected parasites Production of a 6-Cys A,B,E Triple KO 1 4 H G F A B C D E 2 3 I J Days X X A 6-Cys B 6-Cys

22 A triple B. bovis KO line lacking the 6Cys A, B and E genes expressing green and red fluorescent markers

23 Applications of transfected parasites for vaccine development Gene function assignment: functional role of genes (ie: hap2 and the 6-cys family). Genes important for sexual stage development can encode proteins that are subunit vaccine candidates. The triple KO transfected line can be used as a non-transmissible vaccine strain, if attenuated. Because it contain exogenous genes and expresses markers can be easily discriminated from field strains

24 How can we simplify the analysis of sexual stage specific antigens and test the activity of putative blocking antibodies? Sexual stages of Babesia bovis are difficult to study since they occur in the relatively inaccessible environment of the tick midgut. In vitro induction of sexual stages using xanthurenic acid was demonstrated in related hemoparasites (Plasmodium and B. bigemina) This approach was also used to study the pattern of expression of the B. bovis hap2 in wild type and hap2 KO parasites

25 Babesia bovis in vitro sexual stages induction using xanthurenic acid Blood Stages Sexual Stages Gametes Mammalian host 37 C XA 26 C 6-cys A RBC Zygote Fertilization HAP2 Live IFA (Strahlenkörper, or ray bodies) Hussein et al., submitted

26 Babesia bovis induced sexual stages Scanning electron micrographs of B. bovis sexual stages, Scale bar: 2µm Hala Hussein-unpublished

27 Combining in vitro induction of sexual stages and KO production using transfection A hap 2 KO mutant was generated using transfection The patterns of expression of 6-cys and hap2 genes and changes in morphology in in vitro XA induced parasites were compared among wild type and mutant parasites.

28 Transfection plasmid design for generating B. bovis hap2 KO Hussein et al., under review

29 Production of a B. bovis D-hap2 clonal line Hussein et al., under review

30 Expression analysis comparing Wild type vs hap2 KO parasites using RT-PCR Hussein et al., under review

31 Expression analysis comparing Wild type vs hap2 KO parasites using Immunoblots Hussein et al., under review

32 Expression analysis comparing Wild type vs hap2 KO parasites using live immunofluorescence analysis (surface exposure) Hussein et al., under review

33 Morphological changes of B. bovis wild type and hap2 KO parasites developing in induced in vitro cultures Sexual form induction results in the development of extra-erythrocytic parasites with long projections and large round parasite stages. Hap2 KO parasites failed to show such morphological changes Hussein et al., PLoS neglected, 2017

34 Functional analysis of B. bovis HAP2 HAP2 is differentially expressed by B. bovis during its development within R. microplus Surface exposed expression of this protein might be connected to the completion of the B. bovis life cycle during parasite development in the tick midgut. hap2 is unnecessary for parasite development during infection of mammalian host Expression of HAP2 occurs in concurrence with the development of sexual stages upon induction with xanthurenic acid The data suggests a role of hap2 during tick stages of the parasite, and supports HAP2 as a leading candidate for a transmission blocking vaccine against bovine babesiosis. Hussein et al., PLoS neglected, 2017

35 Conclusions Definition of the 6-cys A, B and HAP2 as TB-vaccine candidates in B. bovis Gene function analysis using a transfection approach: identification of the requirement of HAP2 for the formation of in vitro induced sexual stages Feasibility of incorporating a vaccine molecular marker Transgenic Babesia bovis lacking 6-Cys or hap2 genes may be the foundation for non-transmissible live vaccines against bovine babesiosis

36 Current-Future Work Expression of Babesia transmission blocking vaccine candidates in transfected parasites. Further expansion to the use of stably transfected B. bigemina Testing and developing non-transmissible B. bovis vaccine strains Developing B. bovis TBV subunit vaccines based on defined candidates

37 Acknowledgments Heba Alzan Hala Hussein Paul Lacy Jacob Laughery Marta Silva Gina Gallegos Reginaldo Bastos Richard Bishop Brian Cooke Audrey Lau Massaro Uetti Mona Mahmoud Edith Orozco David Herndon Ralph Horn James Allison Melissa Flat Don Knowles Glen Scoles

Primer DNA sequence (5 to 3 )

Supplemental Table 1 Oligonucleotide primers used in the study Primer DNA sequence (5 to 3 ) Plasmodium falciparum 18s forward Plasmodium falciparum 18s reverse Plasmodium falciparum MSP1 forward Plasmodium