TARGETING SUBDOMINANT RHOPTRY-ASSOCIATED PROTEINS OF BABESIA BOVIS IN A SUBUNIT VACCINE TO PROTECT CATTLE AGAINST BOVINE BABESIOSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022541
• Grant No.: 2020-67015-31809
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2019-05375
• Project Start Date: Jul 1, 2020
• Project End Date: Jun 30, 2025
• Grant Year: 2020
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
AGRICULTURAL RESEARCH SERVICE
800 BUCHANAN ST, RM 2020
BERKELEY,CA 94710-1105

Performing Department
US Department of Agriculture

Non Technical Summary
Babesia is a tick borne disease with high impact in cattle industries worldwide. The disease was eradicated from the US by eliminating the tick vector. The recent increased risk for the re-emergence of bovine babesiosis in the US through the US-Mexico border indicates that Babesia bovis continues to be a significant threat to US cattle industry. Live attenuated vaccines against B. bovis are available, but not permitted in the US due to their several limitations. Therefore, development of efficient, new generation vaccines to control B. bovis is a high priority. In the present project we propose to develop a subunit vaccine to protect cattle against clinical signs of acute bovine babesiosis using a previously untested approach based on immunosubdominant antigens. We selected totarget suchsubdominant antigens, thosethat induce poor or immune suppressed responses during natural infections, because they may be more likelyessential for parasite survival. We plan to generate and test a vaccine including two previously characterized antigenically subdominant antigens of known functional relevance. The novel subunit vaccine will be tested in cattle trials.We expect that the vaccinated animals will control parasitemia, show mild signs of infection, and survive challenge with a virulent strain of B. bovis. We predict that the novel subunit vaccine will prime the immune systems of cattle, slowdown parasitemia, and ultimately provide time for the host to mount a protective adaptive immune response targeted to control acute disease. Considering our goal to develop a subunit vaccine to control acute bovine babesiosis, this proposal will take a step forward both the NIFA mission and the AFRI strategic goal to provide Americans access to safe, nutritious, and secure food supply.

Animal Health Component

75%

Research Effort Categories

Basic

25%

Applied

75%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1110	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1110 - Parasitology;

Keywords

babesia bovis

bovine babesiosis

subdominant antigen

subunit vaccine

Goals / Objectives
Babesia bovis is a tick-borne apicomplexa parasite anda causative agent of bovine babesiosis, a disease that causes significant morbidity and mortality in cattle in tropical and subtropical areas of the world.Although bovine babesiosis was erradicated from the US in the past , there is a high risk of re-introductionand vaccines are needed. The goal of this project is to develop a subunit vaccine against Babesia boviscontaining RAP-1 NT/RRA subdominant domains fused with the molecular adjuvant Flic to protect cattle against acute bovine babesiosis. Our overall hypothesis is that a subunit vaccine targeting the NT immunosubdominant segment of RAP-1 and full-length RRA of B. bovis fused with the Flic flagellin molecular adjuvant protects cattle against acute bovine babesiosis. Two specific aims are proposed to test our hypothesis. In Specific Aim (SA) 1, we will express, purify, and test the antigenicity of RAP-1 NT and RRA fused with Flic. Recombinant proteins will be expressed in E. coli, purified using nickel columns, and their antigenicity will be evaluated using sera from B. bovis acutely infected and immune cattle. In SA2 we will evaluate whether a subunit vaccine containing B. bovis RAP-1 NT and full-length RRA fused with the molecular adjuvant Flic protects cattle against clinical signs of acute bovine babesiosis.

Project Methods
For Specific aim #1:1.Cloning and expression of recombinant proteins: E. coli codon-optimized genes coding for the proteins of interest will be synthetically produced, cloned, and expressed into the pBAD/TOPO™ TA vector (Invitrogen) as recommended by the manufacturer. Recombinant proteins will be purified by affinity to a nickel column with ProBond™ resin (Invitrogen) following the manufacturer's protocol.2.Antigenic evaluation of recombinant RAP-1 NT/Flic and RRA/Flic: Antigenicity of recombinant proteins will be evaluated using sera froma B. bovis-infected cattle.Our laboratory has a large collection of serum samples from B. bovis acutely infected and hyperimmune cattle that will be used to assess antigenicity of the target antigens by indirectELISA and immunoblot testings. ELISA will be performed by standard protocol. monoclonal and polyclonal antibodies against RAP-1 and RRA, available in our laboratory, will be used as controls for the immunoblots.For Specific Aim #2:3. Vaccination of cattle with recombinant antigens: Four groups of fourteen 1-year old Holstein calves will receive four subcutaneous inoculations, at 4-week intervals, of recombinant protein emulsified in adjuvant Montanide™ ISA (Seppic). The immunization groups will be as follows: (group 1, n=14) inoculation of 20 μg of RAP-1 NT/Flic; (group 2, n=14) 20 μg of RRA/Flic; (group 3, n=14) 20 μg of RAP-1 NT/Flic plus 20 μg of RRA/Flic; and (group 4, n=14) 23 μg of Flic alone. For inoculation, recombinant proteins will be emulsified in 1 ml of adjuvant Montanide™.Based on the Fisher's Exact Conditional Test and alpha = 0.05 for the power calculation, fourteen animals per group are needed to evaluate protection against challenge with a virulent strain of B. bovis. Considering the number of animals proposed to evaluate the experimental differences among groups, a randomized block design will be used. All animal experiments will be performed in accordance with the Washington State University Institutional Animal Care and Use Committee, the University of Idaho Institutional Animal Care and Use Committee, and USDA Animal Care Regulations.4.. Evaluation of immune response in vaccinated cattle: Starting at 15 days post the first inoculation, sera and peripheral blood mononuclear cells (PBMC) will be collected weekly from vaccinated animals to evaluate humoral and cellular immune responses to the recombinant proteins. RAP-1 NT/Flic and RRA/Flic will be used to develop direct ELISAs to detect antibodies in the vaccinated calves as described in section 2. Anti-RAP-1 monoclonal antibodiesand polyclonal antibodies against RRAwill be used to standardize the respective ELISAs. Pre-vaccination sera and serum samples from the control group will be used to assess the specificity of the RAP-1 NT/Flic and RRA/Flic ELISAs.Humoral response to Flic alone will be assessed by ELISA. The ELISA for the target antigens will bemodifiedto determine the IgG1/IgG2 ratio in vaccinated animals. Sera from the vaccinated animals will also be used for immunoblot, as described in section 2.Sera from the vaccinated cattle will be tested for their ability to inhibit cytoadhesion of B. bovis-infected RBC in vitro, as previously described using cultured bovine brain endothelial cells (BBEC) .Development of neutralizing antibodies in the vaccinated cattle will be evaluated using a flow cytometry-based approach utilizing a GFP-expressing B. bovis strain developed in our laboratories (Fig. 5), as previously described (54). Briefly, GFP-expressing B. bovis will be cultured in a 96-well plate by standard protocol. Incremental dilutions of serum from vaccinated and control animals will be added to the Babesia culture growth medium and the percentage of parasitized RBC (pRBC) will be determined by flow cytometer counting 20,000 events per sample. Percentage of parasitemia inhibition will be measured at days 1 through 4 of culture. The percentages of parasitemia inhibition (% pi) will be calculated with the following formula: % pi = 100 - ([(pRBC Post) / (pRBC Pre)] × 100). Results of parasitemia inhibition in three replicates will be analyzed.Starting at 15 days post the first inoculation, peripheral blood will be collected weekly from vaccinated animals and their PBMC will be used to evaluate cellular immune responses to the recombinant proteins. PBMC will be purified by Histopaque® (Sigma) gradient centrifugation and used for NO production assay, IFNγ ELISpot, IFNγ intracellular staining (ICS), and EdU cell proliferation. Production of NO in antigen-stimulated PBMC will determined by measuring the accumulation of nitrite, the stable oxidized form of NO, in cell culture medium using the Griess reaction. Briefly, PBMC from vaccinated and control cattle will exposed to serial concentration of recombinant antigens for 96 hours, as previously described (55). Recombinant bovine IFNγ (50 U/ml) (Ciba-Geigy) plus recombinant human TNFα (2,500 U/ml) (R&D Systems) will be used as positive controls for NO production. After incubation, 50 µl of cell culture supernatant will be then transferred to a 96-well plate and Griess reaction performed following the manufacture's protocol (Promega). The experiment will be repeated three times, and samples tested in duplicate each time. Results will be shown as the mean micromolar concentration of nitrite in cell culture supernatant.Detection of secreted IFNγ in antigen-stimulated PBMC from vaccinated cattle will be performed using the bovine IFNγ ELISpot kit from MabTech following the manufacturer's instructions.IFNγ ICS will also measuredusing methods established in our labs.based onflow cytometry. To evaluate proliferation in antigen-stimulated PBMC from vaccinated cattle, the Click-iT® EdU Flow Cytometry Assay Kit will be used following the manufacture's protocol.Statistical significance for the proposed immune assays will be determined using a one-way ANOVA with a Tukey correction for multiple comparisons, and results will be considered significant when P <0.05. All the cellular immune assays proposed have been previously optimized by our team and are currently in use in our laboratory. Preliminary results of IFNγ ELISpot, IFNγ ICS, and EdU cell proliferation are shown in Figure 6.5. Challenge and post-challenge evaluation of vaccinated cattle: After the fourth immunization and development of evident immune response to the recombinant proteins, vaccinated and control cattle will be challenged intravenously with 5x104 B. bovis infected RBC (T2Bo virulent strain). This dose of parasites will induce pathognomonic signs of acute bovine babesiosis, including fever and drop in PCV in naïve cattle. Challenged animals will be monitored daily for clinical signs of babesiosis, including fever and drop in PCV. During the first 20 days post-challenge, blood samples will be collected daily from each animal to determine parasitemia. Parasite load in whole peripheral blood will also be tested by qPCR as previously described . Immunological assays described in section2 will be performed weekly after challenge. Data of parasitemia, parasite load in peripheral blood, immune response post-challenge, and clinical signs of acute babesiosis will analyzed to determine the level of protection in vaccinated animals compared to controls. Protection following challenge will be defined as a combination of significant differences in PCV, rectal temperature, parasitemia, parasite load, and survival rate comparing vaccinated and control cattle. Cattle that develop a combination of fever (>40.2°C), inappetence for two consecutive days, and >50% drop in PCV will be humanely euthanized via intravenous injection of Fatal Plus (Vortech Pharmaceuticals, USA) according to the manufacturer's dosing instructions.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:Preliminary data were presented in a poster format at the CRWAD meeting, 2024 (Immunological evaluation of recombinant RAP-1 and RRA proteins of Babesia bovis. Manuel Rojas, Reginaldo G. Bastos, Jinna Hoyos, Jacob M. Laughery,Paul A. Lacy, Carlos E. Suarez). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training a PhD graduate student (Manuel Rojas) How have the results been disseminated to communities of interest?By presenting at scientific meetings (CRWAD 2024) and scientific peer reviewed journals (Frontiers in Immunology and Nature Scientific Reports). What do you plan to do during the next reporting period to accomplish the goals?Weare currently performing a cattle vaccine trial using recombinant RAP-1 NT and RRA with Flic adjuvant. Results and outcomes from these experiments will be included in the next technical report.

Impacts
What was accomplished under these goals? Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily inB. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Sequence comparisons among the RRA sequences of severalB. bovisstrains and otherBabesiaspp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinctBabesiaspecies. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of theB. bovisRRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines againstB. bovis. In addition,conserved antigens expressed in the rhoptries are rational vaccine targets, but whether conservation of protein structure is a functional requirement for invasion remains unknown. Novel protein structural modeling enables identification of structurally conserved protein families that are not evident by sequence analysis alone. We showed by AlphaFold2 structural modeling that the rhoptry-associated protein 1 superfamily of the Piroplasmida hemoparasites Babesia and Theileria (pRAP-1) is structurally conserved, with the core conserved region being composed of a globin-like and a 4-helix bundle subdomain. Search for structurally related members of this protein family in other apicomplexan parasites revealed structural homologues of pRAP-1 in several species of Plasmodium, Toxoplasma gondii and other members of the Sarcocystidae family. Based on these structural findings, pRAP-1 is a conserved apical complex protein, but whether these proteins share functional features in different species remains unknown. Identification of widely conserved elements involved in infection in these parasites will enhance our knowledge of invasion mechanisms, and facilitate the design of methods for controlling diseases that affect humans and animals globally.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Front Immunol. 2024 Apr 24:15:1380660. doi: 10.3389/fimmu.2024.1380660. eCollection 2024.A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.Rojas MJ, Bastos RG, Navas J, Laughery JM, Lacy PA, Suarez CE.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: CRWAD 2024, Proceedings. P057 - Immunological evaluation of recombinant RAP-1 and RRA proteins of Babesia bovis Manuel J. Rojas, Reginaldo G. Bastos, Jinna Navas, Jacob M. Laughery, Paul A. Lacy, Carlos E. Suarez
• Type: Journal Articles Status: Published Year Published: 2023 Citation: H�tzel I, Suarez CE. Structural definition of babesial RAP-1 proteins identifies a novel protein superfamily across Apicomplexa. Sci Rep. 2023 Dec 15;13(1):22330. doi: 10.1038/s41598-023-49532-0. PMID: 38102310; PMCID: PMC10724250.

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:Results of our project will benefit: (a) stakeholders of the cattle industry in the US and worldwide; (b) scientific community involved in researching parasitic diseases and vaccine development; and (c) government regulatory agencies in endemic countries and areas at-risk for bovine babesiosis, such as the US-Mexico border in Texas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate student (Manuel Rojas, PhD student - Immunology and Infectious Diseases, Washington State University - College of Veterinary Medicine, Dept. Veterinary Microbiology and Pathology). Training of technical lab support - Jinna Navas (lab technician; Washington State University - College of Veterinary Medicine, Dept. Veterinary Microbiology and Pathology). How have the results been disseminated to communities of interest?Preliminary data were presented in a poster format at the CRWAD meeting, 2023 (Immunological evaluation of peptides derived from the Babesia bovis RAP-1 and RRA vaccine candidate antigens. Manuel Rojas, Reginaldo G. Bastos, Jinna Hoyos, Jacob M. Laughery,Paul A. Lacy, Carlos E. Suarez). Preliminary data were presented in an oral format at the IBM-IV, 2023 (Early increase in circulating monocytes during Babesia bovis acute infection correlates with protection against bovine babesiosis. Reginaldo G. Bastos, Janaina Capelli-Peixoto, Jacob M. Laughery, Heba F. Alzan, Sezayi Ozubek, Massaro W. Ueti, Carlos E. Suarez). Publications in peer-reviewed scientific journals: Bastos RG, Laughery JM, Ozubek S, Alzan HF, Taus NS, Ueti MW, Suarez CE. Identification of novel immune correlates of protection against acute bovine babesiosis by superinfecting cattle with in vitro culture attenuated and virulent Babesia bovis strains. Front Immunol. 2022 Nov 18;13:1045608. doi: 10.3389/fimmu.2022.1045608. eCollection 2022. Capelli-Peixoto J, Saelao P, Johnson WC, Kappmeyer L, Reif KE, Masterson HE, Taus NS, Suarez CE, Brayton KA, Ueti MW. Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts. Front Cell Infect Microbiol. 2022 Dec 19;12:1093338. doi: 10.3389/fcimb.2022.1093338. eCollection 2022 Johnson WC, Hussein HE, Capelli-Peixoto J, Laughery JM, Taus NS, Suarez CE, Ueti MW. Transfected Babesia bovis Parasite Line Expressing eGFP Is Able to Complete the Full Life Cycle of the Parasite in Mammalian and Tick Hosts. Pathogens. 2022 May 27;11(6):623. doi: 10.3390/pathogens11060623. What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 2: This aim will be performed subsequently to specific aim 1.

Impacts
What was accomplished under these goals? Specific Aim 1: Express, purify, and test the antigenicity of RAP-1 NT and RRA. Major activities completed / experiments conducted: The rational development of recombinant vaccines based on subdominant antigens requires a better understanding of the immune correlates of protection against the disease of interest (e.g., bovine babesiosis). It also requires studying the antigenic profile of the target subdominant antigens, for instance RRA, RAP-1NT and conserved peptides derived from these proteins. To this end, we first decided to use a cattle infection model based on vaccination using attenuated live parasites, followed by challenge with a virulent strain. This model provided us with the necessary information and reagents to identify novel correlates of protection against acute bovine babesiosis caused by B. bovis, and the tools needed for antigenic analysis and subsequent vaccine trials. Findings from this initial study demonstrated novel alterations in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. Another required component of the project is the availability of recombinant RAP-1NT, RRA and Flic proteins. We first expressed the target recombinant proteins RRA, RAP-1 NT in a eukaryotic expression system based on HEK293 cells. However, poor solubility of the target proteins in water-based buffer made us to reevaluate our approach to investigate antigenicity. Given these limitations, we decided to express the two recombinant proteins RRA and RAP-1NT, as well as FliC, in a prokaryotic expression system based on E. coli. As a result, we now have the three recombinant proteins available in sufficient amounts that will allow performing the vaccine trials. Antigenicity of RAP-1NT and RRA was investigated by iELISA and Western blot analysis using conserved peptides and recombinant RRA and RAP-1NT proteins vs. sera from acutely and persistently experimentally infected bovines and rabbits immunized with synthetic peptides. By using this approach, we were able to demonstrate that B. bovis-infected bovine sera have significant less antibodies to RAP-1 NT and RRA recombinant proteins and synthetic peptides than against immunodominant RAP-1 CT peptides. In summary, this experiment revealed critical information on the relationship between the cattle immune system and parasite antigens. Data collected: ELISA and immunoblot analysis were carried out using standard protocols. Synthetic peptides were manufactured by a commercial company following standard procedures. Recombinant Flic, RRA, and RAP-1NT proteins were produced in E. coli and purified using Ni2+ columns. All animal experiments were performed following the guidelines approved by institutional Animal care and use committee (IACUC) of Washington State University and University of Idaho. c. Summary statistics and discussion of results: The experimental cattle infection model demonstrated novel correlates of protection. Briefly, this included monocytosis, neutropenia, and CD4+ lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle and are consistent with the effect demonstrated by the use of FliC as an immunostimulant/adjuvant in cattle studies, as proposed in this project. We also demonstrated that B. bovis-infected cattle develop higher levels of antibodies against immune dominant segments of RAP-CT than against sub-dominant segments RAP-1 NT and RRA. Using these tools, we will also determine the functional relevance of conserved and non-immunodominant RAP-1 and RRA domains.Preliminary structural simulations using alpha fold shows identical and overlapping structural configuration in the RRA and RAP-1NT proteins suggesting possible similar or redundant functions for RAP-1 and RRA. This observation supports the need for the use of both proteins in a successful vaccination scheme based on RAP-1. Altogether, these results also support our premise and previous observation that RRA and RAP-1 NT are sub-immunodominant antigens that are functionally relevant during erythrocyte invasion by the parasite. Data from experiments with the attenuated B. bovis strain demonstrated that vaccinated animals contain antibodies reactive against key RAP-1 and RRA domains and were protected against challenge with a virulent strain. ?

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Bastos RG, Laughery JM, Ozubek S, Alzan HF, Taus NS, Ueti MW, Suarez CE. Identification of novel immune correlates of protection against acute bovine babesiosis by superinfecting cattle with in vitro culture attenuated and virulent Babesia bovis strains. Front Immunol. 2022 Nov 18;13:1045608. doi: 10.3389/fimmu.2022.1045608. eCollection 2022.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Capelli-Peixoto J, Saelao P, Johnson WC, Kappmeyer L, Reif KE, Masterson HE, Taus NS, Suarez CE, Brayton KA, Ueti MW. Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts. Front Cell Infect Microbiol. 2022 Dec 19;12:1093338. doi: 10.3389/fcimb.2022.1093338. eCollection 2022
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Johnson WC, Hussein HE, Capelli-Peixoto J, Laughery JM, Taus NS, Suarez CE, Ueti MW. Transfected Babesia bovis Parasite Line Expressing eGFP Is Able to Complete the Full Life Cycle of the Parasite in Mammalian and Tick Hosts. Pathogens. 2022 May 27;11(6):623. doi: 10.3390/pathogens11060623.

Progress 07/01/21 to 06/30/22

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate student: Manuel Rojas, PhD student - Immunology and Infectious Diseases, Washington State University - College of Veterinary Medicine, Dept. Veterinary Microbiology and Pathology. Training of technical lab support - Jinna Navas (lab technician; Washington State University - College of Veterinary Medicine, Dept. Veterinary Microbiology and Pathology. How have the results been disseminated to communities of interest?Results from this project were presented at the CRWAD (Conference of Research Workers in Animal Diseases) meeting (Chicago, IL, December 4-7, 2021). What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 2:This aim will be performed subsequently to specific aim 1. We will evaluate whether a subunit cocktail vaccine containing recombinant B. bovis RAP-1 NT and full-length recombinant RRA combined with the molecular adjuvant FliC protects cattle against clinical signs of acute bovine babesiosis.

Impacts
What was accomplished under these goals? Babesia bovis is a tick-borne apicomplexan parasite that causes bovine babesiosis, an economically important disease that affects cattle in tropical and subtropical areas of the world. Acute B. bovis infection is characterized by adhesion of infected erythrocytes in the capillaries of the brain, liver, and lungs, among other vital organs, a condition that resembles severe malaria, leading to high mortality. The available live, blood-based, attenuated B. bovis vaccines have several drawbacks, including: (a) variable virulence, especially in naïve adult animals; (b) restrict use for young calves (<1-year old); (c) risk to transmit contaminating blood-borne pathogens; (d) use of several (>20) calves to produce the attenuated vaccine strains; (e) potential risk for the development of lethal acute babesiosis in vaccinated animals; (f) vaccinated animals become carriers/reservoirs of B. bovis and a risk for tick acquisition/transmission; (g) vaccine batch variation with implications on quality control; and (h) need of a cold chain for vaccine distribution and administration. Considering these disadvantages, the live attenuated vaccines against B. bovis remain non-licensed in the US, and development of efficient, sustainable vaccines to control the parasite is a high priority. Previous attempts to develop subunit vaccines to B. bovis using immunodominant antigens failed in developing an effective vaccine so far. The goal of this project is to develop a subunit vaccine using the Rhoptry Associated Protein 1 N-terminal (NT) immunosubdominant segment (RAP-1 NT) and the full-length RAP-1 Related Antigen (RRA) of B. bovis to protect cattle against acute bovine babesiosis. Considering that RAP-1 NT and RRA are subdominant antigens, we intend to use the molecular adjuvant FliC to boost the immune response against the two target antigens. Our overall hypothesis is that a subunit vaccine targeting RAP-1 NT and full-length RRA of B. bovis combined with the FliC protects cattle against acute bovine babesiosis. Two specific aims are proposed to test our hypothesis. In Specific Aim (SA) 1, we plan to express, purify, and test the antigenicity of RAP-1 NT and RRA fused with FliC. In SA2 we will evaluate whether a subunit cocktail vaccine containing recombinant B. bovis RAP-1 NT and full-length recombinant RRA combined with the molecular adjuvant FliC protects cattle against clinical signs of acute bovine babesiosis. Specific Aim 1: Express, purify, and test the antigenicity of RAP-1 NT and RRA. Major activities completed / experiments conducted: We efficiently expressed the target recombinant proteins RRA, RAP-1 NT in a eukaryotic expression system based on HEK293 cells. However, poor solubility of the target proteins in water-based buffer made us to reevaluate our approach to investigate antigenicity. While we are still pursuing to overcome the technical issues and express full-length RAP-1 NT and RRA, we have started investigating their antigenicity using conserved peptides. Antibodies against the synthetic peptides were also generated and will be used to determine whether these conserved regions contain neutralization sensitive epitopes. Testing antigenicity of recombinant RAP-1NT and RRA in SA#1 requires the availability of antibodies generated in cattle against B. bovis parasites. We thus performed a vaccine/challenge experiment in young (<1-y) and adult (>1-y) cattle using homologous strains of B. bovis. Noteworthy, young animals are naturally more resistant to acute bovine babesiosis than adults. By using this approach, we were able to produce preliminary data showing that B. bovis-infected bovine sera have significant less antibodies to RAP-1 NT and RRA peptides than against immunodominant RAP-1 CT peptides. In summary, this experiment revealed critical information on the relationship between the cattle immune system and parasite antigens. For instance, we demonstrated that cattle infected with an attenuated B. bovis Texas strain developed significant levels of antibodies, including IgM, total IgG, IgG1, and IgG2, against the immunodominant segment of RAP-1 CT. In addition, immunoblot analysis using B. bovis culture as antigen showed that animals infected with the attenuated Texas strain primarily respond against three major immunodominant proteins, likely Bv80, MSA-1, and RAP-1. Strikingly, both young and adult cattle exposed to the attenuated B. bovis Texas strain were resistant against challenge with a homologous virulent strain of the parasite. Next, sera from protected animals will be used in antigenic analysis of immune subdominant antigens RAP-1 NT and RRA in comparison to immunodominant RAP-1 CT. Data collected: ELISA and immunoblot analysis were carried out using standard protocols. Synthetic peptides were manufactured by a commercial company following standard procedures. Animal experiments were performed following the guidelines approved by institutional Animal care and use committee (IACUC) of Washington State University and University of Idaho. c. Summary statistics and discussion of results: By using peptides, we produced preliminary data showing that B. bovis-infected cattle develop higher levels of antibodies against immune dominant segments of RAP-CT than against sub-dominant segments RAP-1 CT and RRA. Using these tools, we will also determine the functional relevance of conserved and non-immunodominant RAP-1 and RRA domains. These results support our premise and previous observation that RRA and RAP-1 NT are sub-immunodominant antigens that are functionally relevant during erythrocyte invasion by the parasite. Data from vaccination experiments demonstrated that vaccinated animals contain antibodies reactive against key RAP-1 and RRA domains and were protected against challenge with a virulent strain. Specific Aim 2:This aim will be performed subsequently to specific aim 1.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Babesiosis as a potential threat for bovine production in China. He L, Bastos RG, Sun Y, Hua G, Guan G, Zhao J, Suarez CE. Parasit Vectors. 2021 Sep 7;14(1):460. doi: 10.1186/s13071-021-04948-3.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family. Bastos RG, Thekkiniath J, Ben Mamoun C, Fuller L, Molestina RE, Florin-Christensen M, Schnittger L, Alzan HF, Suarez CE. Pathogens. 2021 Oct 26;10(11):1384. doi: 10.3390/pathogens10111384.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Harnessing Mycobacterium bovis BCG Trained Immunity to Control Human and Bovine Babesiosis. Bastos RG, Alzan HF, Rathinasamy VA, Cooke BM, Dellagostin OA, Barletta RG, Suarez CE. Vaccines (Basel). 2022 Jan 14;10(1):123. doi: 10.3390/vaccines10010123.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Identification of CCp5 and FNPA as Novel Non-canonical Members of the CCp Protein Family in Babesia bovis. Ozubek S, Alzan HF, Bastos RG, Laughery JM, Suarez CE. Front Vet Sci. 2022 Feb 15;9:833183. doi: 10.3389/fvets.2022.833183. eCollection 2022.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Lactate Dehydrogenase as a Potential Therapeutic Drug Target to Control Babesia bigemina. He L, Bastos RG, Yu L, Laughery JM, Suarez CE. Front Cell Infect Microbiol. 2022 Apr 19;12:870852. doi: 10.3389/fcimb.2022.870852. eCollection 2022.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development. Onzere CK, Fry LM, Bishop RP, Da Silva M, Madsen-Bouterse SA, Bastos RG, Knowles DP, Suarez CE. Int J Parasitol. 2022 May;52(6):385-397. doi: 10.1016/j.ijpara.2022.01.004. Epub 2022 Mar 19.

Progress 07/01/20 to 06/30/21

Outputs
Target Audience:Results of our project will benefit: (a) stakeholders of the cattle industry in the US and worldwide; (b) scientific community involved in researching parasitic diseases and vaccine development; and (c) government regulatory agencies in endemic countries and areas at-risk for bovine babesiosis, such as the US-Mexico border in Texas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate student (Manuel Rojas, PhD student - Immunology and Infectious Diseases, Washington State University - College of Veterinary Medicine, Dept. Veterinary Microbiology and Pathology). Training of technical lab support - Jinna Navas (lab technician) How have the results been disseminated to communities of interest?In the form of a Poster presented at the 101th CRWAD meeting, 2020. (Targetting subdominant rhoptry-associated proteins of Babesia bovis in a subunit vaccine to protect cattle against bovine babesiosis). What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will perform additional experiments to verify the purity of target recombinant proteins and examine their antigenicity using sera from cattle recovered from vaccination and challenge, and from experimental infection with virulent B. bovis. These analyses will set the rationale for Specific Aim 2 to vaccinate cattle using RAP-1 NT and RRA, which is the core goal of the current project. Noteworthy, sera from B. bovis infected cattle for the antigenic analysis are already available in our laboratory.

Impacts
What was accomplished under these goals? We efficiently expressed the target recombinant proteins RRA, RAP-1 NT as well as RRA-Flic and RAP-1NT-Flic, and Flic in a eukaryotic expression system based on HEK293 cells. Subsequently, we were able to purify all these target proteins using immunoaffinity. The completion of these experiments will allow us moving forward towards developing the next planned activity described in our research plan, which is to perform antigenic analysis in the target proteins using sera from acutely and chronically B. bovis infected cattle. Briefly, we aim determining whether all target proteins are recognized by antibodies in B. bovis infected and recovered (protected) bovines. We will also test antibody recognition by cattle that was vaccinated with an attenuated strain of B. bovis. To this end, we also conducted a vaccination and challenge experiment involving attenuated and virulent strains of B. bovis. As a result of this activity, we generated immune sera from vaccinated and recovered cattle that can be used in the antigenic analysis described above.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Pursuing effective vaccines against cattle diseases caused by apicomplexan protozoa.Florin-Christensen, M., Schnittger, L., Bastos, R. G., Rathinasamy, V. A., Cooke, B. M., Alzan, H. F., Suarez, C. E. CAB Reviews 2021, 16,No024
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Bovine Babesiosis in Turkey: Impact, Current Gaps, and Opportunities for Intervention. Ozubek S, Bastos RG, Alzan HF, Inci A, Aktas M, Suarez CE.Pathogens. 2020 Dec 11;9(12):1041. doi: 10.3390/pathogens9121041.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Targetting subdominant rhoptry associated proteins of Babesia bovis in a subunit vaccine to protect cattle against bovine babesiosis. Bastos R.G., Taus NS, Suarez CE. 101 CRWAD Meeting, Chicago, IL, 2020.