Source: TEXAS A&M UNIVERSITY submitted to NRP
DEVELOPMENT OF RECOMBINANT ANTIBODIES AS DIAGNOSTIC, PROPHYLACTIC, THERAPEUTIC AND PEST CONTROL MOLECULES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1000631
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Aug 6, 2013
Project End Date
Jul 31, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Veterinary Pathobiology
Non Technical Summary
Antibodies are formed by a vertebrate host in response to antigens residing on or in microbes. Antibodies bind to specific signature molecules (epitopes) on the antigens. Antibodies can be either polyclonal or monoclonal and are extensively used in diagnostic tests for the detection of specific pathogens and in treatment or prevention of diseases. Polyclonal antibodies (pAbs) found in blood and other body secretions are a mixed population of antibodies that bind to multiple epitopes on a single antigen (or many antigens), whereas monoclonal antibodies (mAb) consist of a homogeneous population of antibodies that bind to a single epitope of an antigen. Since antibodies are present in an immune serum (a.k.a anti-serum) from an animal exposed to a particular antigen, serum therapy has been available commercially for more than a century. In fact, passive transfer of antibodies in serum was the only effective treatment and prevention of certain infectious diseases for many years before the discovery of antibiotics. Although advantageous for serum therapy, the presence of cross reactive polyclonal antibodies to several antigens or pathogens poses a problem in specific detection. MAbs developed and produced by hybridoma technology have exquisite specificity for an antigen and have made a remarkable impact in the development of diagnostics and therapeutics especially in targeted drug delivery. However, antibodies prepared in one species for use in a heterologous species have some immunological side effects. There are proponents who do not like the use of live animals for the production of highly concentrated immune sera. Although, mAbs can be prepared by in vitro procedures, these can be expensive and time consuming. Alternatively, antibody engineering allows one time use of blood/tissue cells of an animal to develop a library of recombinant antibodies (rAbs) to the epitopes of an antigen by application of molecular biology techniques. We intend to reduce or prevent colibacillosis in calves by administering orally a rAb that we have developed. Secondly, we will modify a set of rAbs that we have made for more efficient detection of an E. coli strain (EHEC O157:H7) that can cause disease in humans and is transmitted by animals or animal products. Furthermore, we will develop rAbs for passive immunization and use in a diagnostic test of viral diseases of cattle and pigs. Recombinant antibodies expressed in bacteria (or other in vitro host systems) are safe, effective and can be produced cheaply in highly concentrated form. At present, recombinant antibodies are the most rapidly emerging therapeutic and diagnostic proteins.
Animal Health Component
25%
Research Effort Categories
Basic
20%
Applied
25%
Developmental
55%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310109035%
3113510109020%
3113410109030%
3123310109015%
Knowledge Area
312 - External Parasites and Pests of Animals; 311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal; 3510 - Swine, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology;
Goals / Objectives
Our major long-term goals are to (1) develop and produce recombinant antibodies for diagnosis, treatment and prevention several important infectious diseases of cattle and pigs and (2) develop recombinant antibodies that recognize certain antigens of external pests of domestic livestock and as proof of concept, use in control of these specific pests. Objective 1: i. Development of bispecific recombinant antibodies to capture enterohemorrhagic E. coli O157:H7 (EHEC) from faecal samples for subsequent testing using a PCR assay. At present, available tests for this bacteria use either pAbs or mAbs. In our previous work, we have developed two rAbs that separately recognize either the LPS O157 or the flagella antigen H7; and are able to capture EHEC. Our objective is to modify these rAbs to develop bispecific molecules specific for increased efficiency of capture EHEC from fecal/manure samples. In an assay, the captured bacteria can be tested for the presence of shiga-toxin genes using PCR with electrochemical detector allowing real time monitoring. Such an assay will allow managers to identify infected animals and remove them early from the food chain and will be useful for risk management in pre-harvest food safety. ii. Examine the efficacy of recombinant antibodies in protecting neonatal calves from colibacillosis. We have developed a recombinant antibody from a monoclonal antibody that prevents or reduces colibacillosis in calves. We have shown in vitro that this recombinant antibody inhibits the attachment of the enterotoxigenic E. coli (ETEC-F5) to intestinal epithelial cells. Additionally, we have demonstrated that there is a good correlation of protection afforded by this rAb and protection given by the parental mAb in in vivo ligated intestinal loop system; a classical and accepted assay used widely to test enterotoxigenicity of many bacteria or their products. However, experiments in animals are necessary to validate the results obtained by challenging calves with ETEC-F5 after oral delivery of the rAb. The rAb will provide an alternate cheap source for passive immunization of neonatal calves against colibacillosis. Objective 2: i. The objective is to produce reagents that are crucial in the development of serological diagnostics of African Swine Fever Virus (ASFV). We have, in collaboration with Drs. Berghman and Mwangi, developed mAbs that recognize p54 and p62 of ASFV. We intend to use the hybridoma cells producing these mAbs to fish out genes to develop anti-p54 and anti-p62 rAbs for use in a pen-side assay for the virus. This assay will provide a tool for eliminating a highly contagious foreign animal pathogen that causes major economic losses, threatens food security, and limits pig production in affected countries. ii. Develop a recombinant bovine antibody-based prophylactic and therapeutic product that neutralizes in vitro the infectivity of BVDV. BVDV plays an important role in the pathogenesis of bovine respiratory/diarrhea disease complex (BR/DDC) in neonatal calves. A rAb neutralizing the virus when passively delivered to susceptible calves will induce protection to BVDV, and reduce losses to BR/DDC. Objective 3: Our objective is to develop a phage library for subsequent identification and characterization of rAbs to antigens in/on reproductive organs that will reduce the fecundity of ticks on binding. We intend to use Amblyomma americanum as a model pest to identify rAbs to a target antigen in a `proof of concept study'. Characterization of a rAb binding to the reproductive organ antigens may provide a potential molecule to decrease fecundity of ticks, and thus aid in tick control without the use of chemicals. .
Project Methods
1. i) Development of bispecific recombinant antibodies to capture enterohemorrhagic E. coli O157:H7 (EHEC) from faecal samples for subsequent testing using a PCR assay: The bispecific rAb will be assembled using a linker to tandemly tie the anti-O157 or anti-H7 rAb genes by splicing overlap extension in a two-step PCR. The composite gene will be inserted into the vector pCombX for selection of binder phages by biopanning and subsequently for expression of the bispecific rAb. The soluble rAb expressed will be used to test their ability to precipitate their relevant antigen by immunodiffusion, and to agglutinate EHEC bacteria. Finally, increased efficiency in capturing EHEC, in spiked bovine fecal samples, would indicate that mAbs may be replaced by the rAb in commercial available assays. 1. ii) Examine the efficacy of recombinant antibodies in protecting neonatal calves from colibacillosis: We have proven the effectiveness of anti-ETEC-F5 rAb confirmed in the ligated intestinal loop system to reduce the enteropathogenicity produced by ETEC strain B41. This bacterially expressed rAb will be delivered by oral route to prevent colibacillosis in colostrum deprived calves challenged with ETEC strain B41. Clinical status following challenge of calves will be based on the scoring the fecal consistency, degree of clinical dehydration, and degree of clinical depression. Mean clinical scores with either mAb or rAb to be significantly (P< 0.01) lower than those of control calves would demonstrate the utility of anti-ETEC-F5 rAb for passive immunization of neonatal calves. 2. i) Reagents that are crucial in the development of serological diagnostics of African Swine Fever Virus (ASFV): We will make cDNA from the hybridoma cells expressing anti-p54 and anti-p62 recognizing mAbs. The cDNA will be used in a PCR to amplify the genes for making the rAb phage library in pCombX vector. The phage library will be screened for binders using recombinant p54 and p62 antigens. Once the reactivity to ASFV is proven to be authentic, soluble rAb produced by selected clones can be used in the development of a pen-side test. 2. ii) Develop a recombinant bovine antibody-based prophylactic and therapeutic product that neutralizes in vitro the infectivity of BVDV: We have a steer that has been hypeimmunized with BVDV and its sera demonstrates neutralizing activity. We have made cDNA from B cells of this animal for amplifying the genes to make the rAb phage library in pCombX (as above section 2. i) for screening for binders to the BVDV. After selection and characterization, soluble rAb will be tested for neutralization activity. The rAb from clones expressing neutralizing activity can then be tested for passive immunization of calves in approach similar to 1. ii above. 3. Develop a phage library for subsequent identification and characterization of rAbs to antigens in/on reproductive organs of ticks: We will isolate reproductive organs of female A. americanum nymphs for immunization of steers to obtain B cells that produce reactive pAbs. B cells isolated from these steers will be used to make a phage library as above (section 2). The library will be used for screening for binders to antigens on/in reproductive organs and selected clones used to produce soluble rAb in E. coli. Subsequently, rAb will be fed in vitro to adult female ticks to measure fecundity after full engorgement. RAbs inducing reduced egg output and larval hatch will be used to identify and characterize the antigens as potential targets.

Progress 08/06/13 to 07/31/18

Outputs
Target Audience:Research scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? !) ii. 2) i

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sahagun-Ruiz, A., Velazquez, L., Bhaskaran, S., Jay, C., Morales-Salinas, E., Rathore, K., Wagner, G. and Waghela, S., 2015, Reduction of enterotoxin induced fluid accumulation in ileal loops of neonatal calves with anti-F5 fimbriae recombinant antibody. Veterinary Research Communications 39, 229-236.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Development of monoclonal and monospecific polyclonal antibodies to p54 and pp62 of African Swine Fever virus, a USDA listed select agent, using antigen expressed from synthetic genes.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Research Scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Continue with objective 2 to develop a diagnostic tests for ASFV using the mAbs. For objective 3, we have immunized cattle for developing anti-tick antigens and will be dveloping a phage library if we canobtain research funding.

Impacts
What was accomplished under these goals? Part of objective 1) i by performing in vivo experiments. Part of objective 2) i by developing polyclonal and mAbs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sahagun-Ruiz, A., Velazquez, L., Bhaskaran, S., Jay, C., Morales-Salinas, E., Rathore, K., Wagner, G. and Waghela, S., 2015, Reduction of enterotoxin induced fluid accumulation in ileal loops of neonatal calves with anti-F5 fimbriae recombinant antibody. Veterinary Research Communications 39, 229-236


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Provided laboratory instructions on methodologies for molecular biology and immunology to i)two economically disadvantaged undergraduate students and ii) provided guidance to a graduate student. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1. Graduate student has learned the techniques fro monoclonal development, production and purification. He has also learnt all the techniques necessary to fully characterize the antibodies to the functional level. 2. This graduate student is now conversant with the process of developing and producing recombinant antibodies. At present, he is learning the techniques necessary to increase the functional avidity of selected recombinant antibodies. 3. Undergratuate students have learnt molecular and immunological techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? 1. Work on the objective 1 if we can find a graduate student who has full financila support. 2. Continue on developing the recombinant antibody based diagnostic test for ASFV. 3. Continue in the efforts to increase the avidity of a a neutralizing recombinant antibody to BVDV.

Impacts
What was accomplished under these goals? Objective 2: i) we have developed a phage library, using spleen cells from immunized pigs,that express the recombinant anibodies to p54 and p72 antigens and are at present in the process of biopanning to select high avidity clones. ii) we have selected 5 phage clone candidates that are reactive to the epitope of E2 antigen of BVDV that naturally induce neutralizing antibodies. Further characterization is in process.

Publications


    Progress 08/06/13 to 09/30/13

    Outputs
    Target Audience: Nothing Reported Changes/Problems: 1. Allocation of funds for reagens and personnel continuing the research What opportunities for training and professional development has the project provided? An undergraduate student was trained in molecular biology techniques and the process of expression of rAb in E. coli. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Objective 2 is the priority at present and we are in the process of developing a phage library of rAbs that bind to the pathogens causing BR/DDC.

    Impacts
    What was accomplished under these goals? Objective 2: ii) In process of authenticating a rAb against BVDV and testing the functional efficacy of the protein experessed in mammalian cells.

    Publications