BOVINE HUMORAL IMMUNE RESPONSE UTILIZING ULTRALONG CDR3 ANTIBODIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0228819
• Project Start Date: Mar 27, 2012
• Project End Date: Sep 30, 2018
• 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
There are no therapeutic treatments available for the shrimp viruses that plague the industry, including white spot syndrome, Taura syndrome, and yellowhead disease. There has been some success in the development of specific pathogen resistant stocks, but we do not understand what the genetic basis of those small improvements is. Traditional genetic evaluations for growth and pond survivability employing phenotypic records have been used in several breeding programs [18-20]. This approach has been successful in improving performance traits with high heritabilities, but it is less suitable for lower heritability traits like disease resistance [21]. The immediate expected outcome of this work is the development of a needed reference genome and gene annotation of the Pacific white shrimp that will facilitate future genotype/phenotype studies of farmed shrimp. Furthermore, the expertise needed to implement these bioinformatic tools will be gained by student and professional bioinformaticians working in the principal investigators' laboratories and at the AgriLife Genomics and Bioinformatics Core. This project will serve as a valuable resource for future studies of non-model organisms lacking a reference genome and gene annotation. The potential impact is large for shrimp genomics and the farmed shrimp industry, which has undergone tremendous growth over the past twenty years. The United States is the major consumer of this $9 billion dollar annual industry, yet Asia produces 75% of farmed shrimp. Research is currently underway at our collaborators at Texas AgriLife Research and Texas A&M University in Port Aransas to develop higher density shrimp farming practices [22], which have the potential to shift a larger percentage of farmed shrimp production to the United States. Selective breeding of disease resistant genotypes, highly prolific shrimp brood stocks and stocks with desired traits may be possible through genotype/phenotype studies facilitated by a reference genome and gene annotation. Therefore, the value of generating these genomic resources is substantial.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3721	1080	30%
303	3199	1090	50%
305	3721	1090	20%

Knowledge Area
303 - Genetic Improvement of Animals; 305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3721 - Marine shrimp; 3199 - Invertebrates, general/other;

Field Of Science
1090 - Immunology; 1080 - Genetics;

Keywords

shrimp

aquaculture

immunology

genomics

genetics

Goals / Objectives
In recent years viruses have emerged as the major impediment to successful shrimp farming here and abroad [1, 2]. An immunogenetic approach to pathogen resistance is needed to push this industry into higher density, environmentally friendlier, smaller footprint operations without frequent devastating viral crashes. We have attracted seed monies for reagents and services to sequence the 2.1 gigabase genome [3] of the Pacific white shrimp at the AgriLife Genomics and Bioinformatics Core here at Texas A&M University. The goal of this study is to facilitate assembly and annotation of a reference genome of Litopenaeus vannamei. This crucial tool will bolster competitive proposals to USDA and NOAA/Sea Grant to fund work applying the discovered genetics into breeding better virus resistant shrimp lines. Our implementation of this study will rely primarily on sequencing and bioinformatic services at the Borlaug Center. We will complete this well-defined goal within the temporal and fiscal constraints of this funding mechanism through the following specific objective: Objective 1) Generate a comprehensive transcriptome annotation of protein and RNA coding genes of the shrimp genome using the Cufflinks assembly program. As the world moves towards healthier food sources with less impact on land, water and wild fisheries resources, closed system shrimp mariculture is primed for more years of rapid expansion. Contemporary genomic resources will allow informed immunogenetic selection for viral resistance, even in the high densities required by closed recirculating aquatic support systems.

Project Methods
We will use genomic sequence data derived from massively parallel sequencing (Illumina Genome Analyzer, AgriLife Genomics and Bioinformatics Core) to generate a reference genome of the Pacific white shrimp. We have already begun sequencing. We will use the ALLPATHS-LG assembly program to perform a de novo assembly of this shrimp genome [29]. Tissues have been frozen in RNAlater aqueous preservative as well as Tri-reagent from the individual shrimp being used for the genomic DNA sequencing. This animal (and others) was collected at Bahia de Kino, Gulf of California in the fall of 2011. Poly-A messages will be enriched from total RNA processed from several tissues of this shrimp, including gills, hepatopancreas, brain, heart and lymphoid organ. In Objective 1, we will use cDNA generated from the poly-A enriched RNA to amplify libraries for 76 base paired end sequencing on the Illumina platform. The heterozygosity inherent in the wild animal analyzed will yield substantial SNP data to add to that available in public repositories. We will use the Cufflinks (http://cufflinks.cbcb.umd.edu/) transcriptome assembly program to map reads to the reference genome, to identify polyadenylated and microRNA transcripts, to estimate abundance, and to annotate genes [30].

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Comparative immunologists, vaccine designers, immunogeneticists. 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?Yes, publication in peer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?Experiments will continue as described in the objectives.

Impacts
What was accomplished under these goals? New de novo transcriptome assembly and annotation methods provide an incredible opportunity to study the transcriptome of organisms that lack an assembled and annotated genome. There are currently a number of de novo transcriptome assembly methods, but it has been difficult to evaluate the quality of these assemblies. In order to assess the quality of the transcriptome assemblies, we composed a workflow of multiple quality check measurements that in combination provide a clear evaluation of the assembly performance. We presented novel transcriptome assemblies and functional annotations for Pacific whiteleg shrimp (Litopenaeus vannamei), a mariculture species with great national and international interest, and no solid transcriptome/genome reference. We examined Pacific whiteleg transcriptome assemblies via multiple metrics, and provide an improved gene annotation. Our investigations show that assessing the quality of an assembly purely based on the assembler's statistical measurements can be misleading; we propose a hybrid approach that consists of statistical quality checks and further biological-based evaluations.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Haakenson, J.K., T.C. Deiss1, G.F. Warner, W. Mwangi, M.F. Criscitiello, V.V. Smider. A broad role for cysteines in bovine antibody diversity. Immunohorizons 3(10) 478-487, 2019. PMID: 31619454 DOI: 10.4049/immunohorizons.1900058

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Biomedical scientists, antibody engineers, vaccine developers and bovine immunologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student was trained on this project. Thad Deiss is planning on defending his PhD this fall. How have the results been disseminated to communities of interest?Presentations at immunology and genetics meeeting and peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?Continue immunizing and analyzing bovine antibody repertoires.

Impacts
What was accomplished under these goals? No immunogen to date has reliably elicited broadly neutralizing antibodies (bnAbs) to HIV in humans or animal models. Recent advances in the design of immunogens (BG505 SOSIP) that antigenically mimic the HIV envelope glycoprotein (Env) have improved the elicitation of potent isolate-specific Ab responses in rabbits and macaques, but so far failed to induce bnAbs. One possible contributor to this failure is that the relevant antibody repertoires are poorly suited to target somewhat occluded conserved epitope regions on Env relative to exposed variable epitope regions. To test this hypothesis in a species with a potentially more favorable repertoire, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach nearly 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in the rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and peak breadth (100%, n = 12 global isolates panel) at 217 days. A monoclonal antibody (mAb) isolated from this cow harbored an ultralong HCDR3 of 60 amino acids and neutralized 72% of a large panel of global isolates (n = 117) with a potent median IC50 of 0.028 μg/ml. The mAb epitope mapped to the CD4 binding site of HIV Env, but the binding mode was distinct from known CD4bs antibodies. Thus, despite the inherent difficulty of eliciting bnAb responses to HIV in most test animals, immunization with a trimer mimic in cows was able to generate rapid responses in only 42 days, supporting the notion that the frequency of long HCDR3s is a critical factor in the ability to elicit HIV bnAbs. The results further suggest that immunization of cows may provide an avenue to quickly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Sok, D., K.M. Le, M. Vadnais, K. Saye-Francisco, J.G. Jardine, J. Torres, Z.T. Berndsen, L. Kong, R. Stanfield, J. Ruiz, A. Ramos, C.H. Liang, P.L. Chen3, M.F. Criscitiello, W. Mwangi, I.A. Wilson, A.B. Ward, V.V. Smider and D.R. Burton. Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows. Nature 548(7665):108-111, 2017. PMID: 28726771

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

Outputs
Target Audience:Talks in Mexico and Brazil Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trainees mentored at all levels. How have the results been disseminated to communities of interest?yes, see publication. What do you plan to do during the next reporting period to accomplish the goals?Continuing to other model species platforms, seeking more funding.

Impacts
What was accomplished under these goals? done

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Novel transcriptome assembly and improved annotation of the whiteleg shrimp (Litopenaeus vannamei), a dominant crustacean in global seafood mariculture

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

Outputs
Target Audience: Published paper and spoke in three countries. Changes/Problems: As our competitiors have also found, the repetitive nature of the shrimp genome is an obstacle to good genome assembly. What opportunities for training and professional development has the project provided? yes, many students on project in US and Mexico. How have the results been disseminated to communities of interest? Yes, published in Nature Publishing Group Scientific Reports. What do you plan to do during the next reporting period to accomplish the goals? Stay the course.

Impacts
What was accomplished under these goals? Transcriptome assembly and annotation.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ghaffari, N., A. Sanchez-Flores, R. Doan, K.D. Garcia-Orozco, P.L. Chen, A. Ochoa-Leyva, A.A. Lopez-Zavala, J.S. Carrasco, C. Hong, L.G. Brieba, E. Rudi�o-Pi�ra, P.D. Blood, J.E. Sawyer, C.D. Johnson, S.V. Dindot, R.R. Sotelo-Mundo and M.F. Criscitiello. Novel transcriptome assembly and improved annotation of the whiteleg shrimp (Litopenaeus vannamei), a dominant crustacean in global seafood mariculture. Scientific Reports 4:7081, 2014. View PDF

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: A paper was published in the journal of Bioenergetics and Biomembranes to the target scientific audience, and a presentation was madeat a shrimp mariculture syposium in Hermosillo Mexico. Changes/Problems: Genome assembly has been a challenge. What opportunities for training and professional development has the project provided? We have ample data in this genome and transcrptome for many future students to collaborate in analysis. How have the results been disseminated to communities of interest? A publication and a manuscript in preparation. What do you plan to do during the next reporting period to accomplish the goals? Continue genomic work, publish transcriptome paper.

Impacts
What was accomplished under these goals? Generated a comprehensive transcriptome annotation of protein and RNA coding genes of the shrimp genome using the Cufflinks assembly program.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: L�pez-Zavala, A.A., J.S. Carrasco-Miranda, K.D. Garcia-Orozco, R. Sugich-Miranda, J.M. Hernandez-Flores, M.F. Criscitiello, Luis G. Brieba, Rogerio R. Sotelo-Mundo and Enrique Rudi�o-Pi�era. Crystal structure of shrimp arginine kinase in binary complex with arginine - a molecular view of the phosphagen precursor binding to the enzyme. Journal of Bioenergetics and Biomembranes 45:511-518, 2013.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We have considerable traction on the first objective with a hybrid sequencing approach, including massively parallel sequencing of short fragment, 3KB mate paired and 10KB mate paired libraries to generate whole-genome sequence for assembly. Completed RNAseq will facilitate annotation for the white shrimp genome. With this annotated draft reference genome we will move to the identification of markers (SNP identification) in Objective 2. We will take advantage of the large genetic diversity in wild and commercial broodstock. We predict that in this species there is substantial yet unheralded room for genetic selection and improvement for disease resistance, unlike in species that have been bred domestically for hundreds of years (such as cattle or poultry). PARTICIPANTS: One technician, two students, members of Agrilife Bioinformatics and Genomics Center. TARGET AUDIENCES: Shrimp mariculture researchers and producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The immediate expected outcome is the development of sorely needed genomic and immunogenetic tools for the farmed shrimp industry. The potential impact is large for a booming industry still with tremendous growth potential. The value of farmed Penaeid shrimp worldwide was estimated by the Food and Agriculture Organization of the United Nations at $14.6 billion (www.fau.org/figis). The United States is the major consumer of this industry, yet Asia produces 75% of farmed shrimp. This is a unique opportunity for agricultural genomics to be applied to an animal innate immune system, and we are well poised to deliver the tools to produce more resistant shrimp to provide this high protein food source consistently and with less risk to the growing market.

Publications

• No publications reported this period