US 2008O107652A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0107652 A1 Durvasula et al. (43) Pub. Date: May 8, 2008

(54) METHODS AND COMPOSITIONS FOR Publication Classification CONTROL OF DISEASE IN AQUACULTURE (51) Int. Cl. (75) Inventors: Ravi Durvasula, Albuquerque, NM A6II 35/66 (2006.01) (US); Subba Durvasula, Darthmouth A6IR 39/00 (2006.01) (CA) A 6LX 39/395 (2006.01) A6II 3 L/70 (2006.01) Correspondence Address: A6IP 43/00 (2006.01) LAW OFFICES OF KHALILIANSIRA, LLC 91OOPERSIMMON TREE ROAD (52) U.S. Cl...... 424/135.1; 424/93.2; 424/184.1; POTOMAC, MD 20854 (US) 514/44; 424/164.1; 424/159.1 (73) Assignee: SCIENCE & TECHNOLOGY COR PORATION UNIVERSITY OF NEW (57) ABSTRACT MEXICO STCUNMI Appl. No.: 11/893,699 The invention discloses paratransgenesis methods for preven (21) tion, amelioration or treatment of a disease or disorder in an Filed: Aug. 17, 2007 aquatic animal. The method comprises providing a geneti (22) cally modified microalgae that expresses a recombinant mol Related U.S. Application Data ecule that specifically targets one or more key epitopes of a pathogen that infects the aquatic animal and ii) feeding the (60) Provisional application No. 60/840,278, filed on Aug. aquatic animal directly or indirectly with the genetically 25, 2006. modified unicellular algae.

SCHEMATIC DEMONSTRATING THE PROCESS OF BOAMPLIFICATION

SHUTTLE PLASMD EXPRESSING ANTMCROBAL MOLECULE

S. Q TRANSFORM DUNALELAWITH SHUTLE PLASMD EXPRESSING ANTMICROBAL PROTEIN

TRANSGENCDUNALELLA. GOD X PROTEIN MOLECULES

TRANSGENIC DUNALELLA FED TO ARTEMIA

PARATRANSGENIC ARTEMIA: 1OOOxPROTEIN MOLECULES

PARATRANSGENCARTEMIAFED

TOSHRMPLARWAL STAGES

SHRIMP: 1 OOOOOx PROTEIN MOLECULES

Patent Application Publication May 8, 2008 Sheet 1 of 2 US 2008/0107652 A1

SCHEMATIC DEMONSTRATING THE PROCESS OF BOAMPLIFICATION

SHUTTLE PLASMD EXPRESSING ANTMICROBIAL MOLECULE

TRANSFORM DUNALELLA WITH SHUTTLE PLASMD EXPRESSING ANTIMICROBIAL PROTEIN

TRANSGENC DUNALELLA: GCD X PROTEIN MOLECULES

TRANSGENIC DUNALELLA FED TO ARTEMIA

PARATRANSGENIC ARTEMIA: 1000x PROTEIN MOLECULES

PARATRANSGENIC ARTEMIA FED TO SHRMPLARVAL STAGES

SHRMP: 1 OOOOOX PROTEIN MOLECULES

Patent Application Publication May 8, 2008 Sheet 2 of 2 US 2008/0107652 A1

US 2008/0107652 A1 May 8, 2008

METHODS AND COMPOSITIONS FOR CONTROL approved for shrimp aquaculture in the U.S. Oxytetracycline OF DISEASE IN AQUACULTURE (OTC) and Romet-30 are two antibacterials currently approved in the U.S. for catfish and salmonid aquaculture. CROSS REFERENCE TO RELATED Several combative methods based on drugs are administered. APPLICATIONS Included in this are naturotherapy (latex from Swallowwort, neemcake), chemotherapy (chlorine, OZone, iodine and for 0001. This application claims benefit of priority to the malin). Antibiotics such as Chlorapmphenicol, Oxytetracy provisional patent application No. 60/840.278, filed on Aug. cline, Tetracycline, Ampicillin, Bacitracin, Gentamycin, 25, 2006, the entire contents of which are incorporated herein Neomycin, Streptomycin, Penicillin G, Polymixin-B and by reference. Sulphadiazine are routinely used at shrimp aquaculture facili GOVERNMENT FUNDING ties outside of the U.S. (Parket al., 1994). These have limited Success due to evolution of pathogen resistance to antibiotics. 0002 The present invention was made in part with salary support from the Veteran’s Administration. 0007 Biotechnological approaches hold a promise in the prevention, control and management of disease and disorders I. FIELD OF THE INVENTION associated with marine culture. One approach is to use geneti cally transformed strains of a mariculture that are resistant to 0003. The invention relates to methods and compositions pathogen invasion. For example, procedures for germ line for the control of infections in aquaculture. In particular, the transformation of shrimp have been successfully established invention relates to methods for the control of infections in at UMBI, Center of Marine Biotechnology, Baltimore, Md., commercial aquaculture by paratransgenesis. in France at IFREMER and in Australia at CSIRO. Here the researches have worked on introducing DNA into shrimp by II. BACKGROUND OF THE INVENTION transfection and followed expression and integration of the 0004 World aquaculture production has increased to 59.4 introduced DNA in the host. These procedures can be million metric tons (MT) in 2004, with a value of S70 billion. exploited to produce pathogen-resistant shrimp. Of this, farmed shrimp production accounts for 2.4 million 0008 Whereas germline transformation of mariculture MT, representing a value of nearly S10 billion (FAO 2004). holds promise, issues remain regarding the role of genetically Diseases caused by agents such as White Spot Syndrome modified organisms as human food. Furthermore, stability of Virus (WSSV) and Vibrio species have decimated shrimp germline transformation and viability of genetically modified farming industries in many parts of Asia and South America, offspring may present challenges. and account for nearly $3 billion of economic loss annually. Unregulated use of antibiotics in farmed shrimp and fish 0009. The application of transgenic technologies to operations has widely been banned and contributes to the marine and freshwater algae, diatoms and cyanobacteria is a epidemic of drug-resistant bacteria in humans. Intensive new and rapidly evolving field. Whereas the genetic compo practices that involve meticulous water exchange with strict sition of some of these organisms is well characterized, appli standards of hygiene have been effective in reducing trans cation of recombinant DNA technologies to generate biologi mission of infectious pathogens in farmed shrimp (Otoshi et cally enhanced or augmented forms is at a nascent stage. The al. 2001 and 2002), but are impractical in many lower-income expression of foreign, biologically active molecules by settings of the world. Usually, appearance of disease is asso genetically modified algae offers great potential for large ciated with loss of harvest for shrimp farmers which accounts scale and economical production of many proteins of com for the loss of 30% of global production. The economic mercial and therapeutic significance. impact of infectious diseases of mariculture is overshadowed 0010 Several reports indicate that algae such as Chlamy only by their tremendous threat to global food security. domonas reinhardtii (Mayfield 2003) and Phaeodactylum 0005 The United States is the second largest importer of tricornutum (Zaslavskaia 2001) may be genetically manipu shrimp in the world. Shrimp aquaculture, like other animal lated to express heterologous proteins. Using a chloroplast husbandry industries, is subject to disease, especially under transformation system, Mayfield et al. demonstrated expres current intensive farming methods. In the USA, more than 50 sion of a functional large single-chain (lsc) antibody in C. diseases are associated with aquaculture operations and affect reinhardtii. The antibody, directed against glycoprotein D of shellfish and fish. For example in the shrimp industry, dis human herpes simplex virus, was produced in Solubilized eases are associated with parasites (70%), bacteria (27%) and form by the alga and assembled into higher order complexes fungi (3%) caused by about 20 pathogens including the gas in vivo. In an earlier study, Zaslavskaia et al. engineered P tero-intestinal Vibrio harveyi, V parahaemolyticus and V tricornutum with either a human (glut 1) or Chlorella (hup 1) Vulnificus. It is of interest to note a) of the nine known patho glucose transporter gene. The resulting conversion of a pho genic strains of Vibrio, five are common to humans, and b) tosynthetic autotroph to a heterotroph capable of obtaining natural assemblages of algae live in association with several exogenous glucose in the absence of light energy was a sig species of bacteria and viruses. nificant advance in algal biotechnology. 0006 To prevent the diseases, it is crucial to understand 0011. Of the total 40,000 species of micro algae, 4500 are the functioning of the pathogens, how they affect the com marine species of which 250 are known to grow rapidly mercially important high-density stressed mariculture opera leading to either seasonal or atypical bloom formation. A tions, and how the marine animal would fight the disease. The study of the blooms is important not only for their contribu rapid growth of this industry has outpaced efforts by research tion to trophodynamics of the ecosystem but also due to mass ers, pharmaceutical companies, and federal regulatory agen mortalities of several biota associated with anoxic conditions cies to provide approved therapeutics for disease manage resulting from disintegrating organic mass. There are ment of marine. Currently, there are no antibacterials approximately hundred micro-algae that produce specific US 2008/0107652 A1 May 8, 2008

toxins (Fogg 2002). Of these, about 60 dinoflagellates are model, stable nuclear transformation of V. Carteri was known to cause red tides and some produce toxins causing reported in 1994 by Schiedlmeier et al. Elegant studies with Diaetic Shellfish Poisoning (DSP), Paralytic Shellfish Poi the intent of generating selectable markers for gene replace soning (PSP), Neurotoxin poisoning (NSP) and Ciguatera. ment and gene disruption analysis were Subsequently devel Because some of these algae are consumed either as food or oped (Hallmann and Sumper 1994). One of these studies passively filtered and retained by the commercially important resulted in a V. Carteri transformant that carried the Chlorella shellfish, bioaccumulation of toxins takes place in the marine hexose/H" symporter that is able to survive in the presence of food web. glucose in the dark (Hallmann and Sumper 1996). As in the case with diatoms, this development will only accelerate the 0012 Red tide organisms are known to cause severe eco development of commercial expression systems for V cart nomic losses and set backs to human health. Globally the economic losses could be as high as USS 20 billion and 3.5 to el. 7 million disability adjusted life-years, much in excess of 0016. In all these investigations the most important objec those caused by Chagas disease (GESAMP 2001) and are tive was the stable integration of the transgenes, their autono comparable to those caused by epidemics such as malaria, mous replication and proper expression of the gene product. and diabetes. Additionally, bacterial and viral contamination It is imperative that in mariculture operations the nutritional of the water may cause considerable mortality to larvae of content of the genetically modified alga is not significantly commercially important species. altered and the product is quite similar to that of a non genetically modified organism. 0013 Marine diatoms have also been investigated for genetic Studies. Only a few marine diatoms Skeletonema cos 0017 Marine Cyanobacteria or the bluegreens are ubiq tatum, Cyclotella cryptica, Navicula saprophila and Phaeo uitous and in the open ocean account for 50% of photosyn dactylum tricornutum are so far utilized in gene transforma thetic production (Platt, Subba Rao and Irwin 1983). tion studies. Smith and Alberte (1995) have succeeded in Although Cyanobacteria are more exacting in their growth transferring the animal virus SV40 or plant virus CaMV35S requirements, they are amenable to culture under laboratory promoters into a marine diatom Skeletonema costatum. conditions. Besides feeding commercially important animals Although no stable integration was achieved, B-glucoron Such as shellfish and larvae, these cultures find applications in idase and luciferase have been expressed in S. costatum natural products such as pigments, pharmaceuticals, fatty reporter genes. More recently, using particle bombardment acids, polysaccharides, wastewater treatment, and biodegra technique on diatom cultures, where a high pressure helium dation of pollutants (Elhai 1994). pulse delivers nucleic acids, Dunahay et al (1995) in Cyclo tella cryptica, Navicula saprophila and Apt et al (1996) and 0018. The green alga Chlamydomonas reinhardtii has Zaslavskaia et al (2000) in Phaeodactylum tricornutum suc long served as a model system for photosynthesis and flagel ceeded stable transformation of DNA. Further, Zaslavskaia et lar. This unicellular green alga will grow on a simple medium all (2001) have successfully genetically engineered and con of inorganic salts in the light, using a photosynthesis system Verted a photosynthetic diatom Phaeodatylum tricornutum to that is similar to that of higher plants to provide energy. grow on exogenous glucose in the dark. Chlamydomonas will also grow in total darkness if an alter nate carbon Source, usually in the form of acetate, is provided. 0014. The bottleneck for genetic transformation of dia Both the ~15.8 Kb mitochondrial genome (Genbank acces toms was resolved in 1995. Dunahay et al generated lines of sion: NC001638 (Vahrenholz et al. 1993)) and the complete transgenic Cyclotella cryptica and Navicula saprophila with >200 Kb chloroplast genome for this organism are available plasmid vectors containing the E. coli neomycin phosphtrans online (Genbank accession: BK000554 (Maulet al. 2002)). ferase II gene using helium accelerated particle bombardment The current assembly of the nuclear genome is available (Dunahay et al. 1995). This was followed by the successful online at http://genomegi-psf.org/Chlre3/Chlre3.info.html. transformation of Phaeodactylum tricornutum (Apt et al. The Chlamydomonas Center located at www.chlamy.org 1996) and Cylindrotheca fisiformis (Fisher et al. 1999). A continues to be an informative resource to the Chlamydomo landmark transformation study was demonstrated by nas community. Zaslavskaia et al in 200 r. Most diatoms are solely photosyn thetic and lack the ability to grow in the absence of light. 0019 Dunaliella is a unicellular, bi-flagellated green alga These investigators successfully engineered P tricornutum, a that belongs to the class Chlorophyceae. Morphologically, photosynthetic diatom, to grow on exogenous glucose in the Dunaliella is very similar to Chlamydomonas. Both organ dark by transformation with the glucose transporter gene isms have complex life cycles that encompass, in addition to Glut 1 from human erythrocytes or Hup 1 from the microalga division of motile vegetative cells, the possibility of sexual Chlorella kessleri. Positive transformants exhibited glucose reproduction. These organisms are both photosynthetic, and uptake and grew in the dark in the presence of glucose relatively easy to maintain in a laboratory setting. Unlike (Zaslavskaia et al. 2001). The exciting trophic conversion of Chlamydomonas, the genetics of Dunaliella are poorly an obligate photoautotrophic diatom is a critical first step for understood. Dunaliella is by far one of the most salt-tolerant Successful large-scale cultivation using microbial fermenta eukaryotic organisms (Ben-Amotz and Avron 1990). Further tion technology. Commercial benefits from Such a system are more, it is highly resistant to stresses such as high light enormous, ranging from an increase in biomass and produc intensity and dramatic pH and temperature changes. tivity to reduced loss from contamination by obligate photo Although there is an increasing interest in the mechanisms tropic microbes. that allow Such physiological versatility, research in this area is still in its infancy. To date, few of these stressed-induced 0.015 The multicellular organism Volvox carteri repre genes have been cloned from Dunaliella (Fisher et al. 1996; sents an ideal model organism to study the transition from Fisher et al. 1997: Sanchez-Estudillo et al. 2006), and the unicellularity to multicellularity. Using C. reinhardtii as a information that is available has shed little light on the US 2008/0107652 A1 May 8, 2008

genomic organization or the biological significance of some baum et al. 2002). After intramuscular injection of plasmid of the unique sequence features that have been identified (Sun DNA carrying promoter-driven reporter genes, protein et al. 2006a). expression has been achieved in common carp (Hansen et al. 0020. One of the stress-induced responses in Dunaliella is 1991), tilapia (Rahman and Maclean 1992), goldfish (Kanel the production and accumulation of the carotenoid, 0-caro los et al. 1999), Zebrafish (Heppel et al. 1998), Japanese tene. Dunaliella is one of the richest natural producers of flounder (Takano et al. 2004) and gilthead seabream (Verriet carotenoid, producing up to 15% of its dry weight under al. 2003). Suitable conditions. Interestingly, it is thought that the caro 0023 Although there are several ways to administer vac tenoid functions as a “Sun-screen” to protect chlorophyll and cines, most young fish continue to be vaccinated by hand. In DNA from harmful UV-irradiation (Ben-Amotz et al. 1989). Norway, for example, over 200 million fish are vaccinated The commercial cultivation of Dunaliella began in the 1960s each year. Each fish is removed from the water, anesthetized once it was realized that their halotolerance allowed for and vaccinated. This method is highly stressful for the fish, monoculture in large brine ponds. The ease of maintaining and in Some circumstances rather impractical. Another Dunaliella in culture, its ability to grow in very high salt method of vaccination is by dip immersion into a solution concentrations, tolerance to high temperature and to extreme containing the vaccine. Dip immersion is usually used in fish pH changes, makes this species a highly desirable target for stocks that are too young or Small for manual handling. exploitation as a biological factory for the large-scale produc Unfortunately, this method alone is not sufficient to achieve a tion of foreign proteins. long duration of protection. Thus, the fish are usually Sub 0021 Although the genetics of Dunaliella are poorly jected to intraperitoneal re-vaccination injection as soon as understood, this organism is highly Suited as an algal biore their size allows. Oral vaccine delivery systems are by far the actor. It can be cultured easily, rapidly and inexpensively. most desirable method for immunizing fish. But reports have Until recently, the use of Dunaliella was limited by the indicated that this system is ineffective. All these hurdles absence of an efficient and stable transformation system. The point to the need for the development of a more user-friendly first report of Successful manipulation of D. Salina was by methodology for vaccine administration (Lin et al. 2005). Geng etal. in 2003. Using electroporation, these investigators White Spot Syndrome Virus (WSSV) were able to generate stable transformants carrying the hepa titis B surface antigen. Walker et al. in 2005 reported the 0024 WSSV is the most striking example of shrimp viral isolation and characterization of two D. tertiolecta nuclear disease. This disease has devastated many parts of the world RbcS genes and their corresponding 5' and 3' regulatory with grave economic consequences and reduction in available sequences. The functionality of these regulatory regions was food supply. Infection of peneaid shrimp by WSSV can result initially used to drive the expression of a selectable marker in in up to 100% mortality within 3 to 7 days. The virus is C. reinhardtii. Subsequently, this expression cassette was extremely virulent and has a broad host range including other electroporated into Dunaliella where both stable and tran marine invertebrates such as crayfish and crab. The global sient transformants expressing the ble resistance gene were annual economic loss due to WSSV is estimated to be S3 isolated. Jiang et al. (2005) identified and later used the 5' billion (Hill 2005). In much of the world, there is currently no flanking region of an actingene from D. Salina to direct stable effective method to control this disease. expression of the bialaphos resistance gene (bar) in D. Salina. 0025 Entry and pathogenesis of WSSV in peneaid shrimp In more recent work, Sun et al. (2006b) introduced a func occur either via oral ingestion or water-borne contact (Chou tional nitrate reductase gene into a D. Salina mutant that et al. 1998). Work by several investigators has demonstrated lacked the gene. This group showed that the introduced gene that VP28, a structural protein found on the virion envelope, was able to complement the nitrate reductase defective is responsible for viral attachment, penetration and conse mutant of D. viridis. All the studies described are pivotal to quently the systemic infection of shrimp (Qhappeletal. 2004: the development of an effective transformation system in van Hulten et al. 2001). Although studies on the shrimp Dunaliella, opening the door for the use of this alga as a immune response are limited, the presence of viral inhibiting bioreactor for production of recombinant proteins. proteins in both experimental and natural survivors of WSSV 0022. In recent years, vaccines based on recombinant infections suggests that an adaptive immune response exists DNA technology appear to be a promising approach to con (Venegas et al. 2000: Wu et al. 2002). Several approaches trolling infectious diseases in farmed fish (Biering et al. 2005; using VP28 and another structural envelope protein, VP19, Clark and Cassidy-Hanley 2005; Heppel et al. 1998). By have been used to elicit an immune response in shrimp. Wit intramuscular injection of eukaryotic expression vectors teveldt et al. (2004) orally vaccinated P. monodon and L. encoding the sequence of a pathogen antigen, DNA vaccines vannamei (Witteveldt et al. 2006), two of the most important offer a method of immunization that overcomes many of the cultured shrimp species, using feed pellets coated with inac disadvantages such as risk of infection and high costs of tivated bacteria that were over-expressing VP28. In both traditional live attenuates, killed or subunit protein-based cases, lower mortality was found in test versus control ani counterparts. They induce strong and long-lasting humoral mals up to three weeks post vaccination. In a similar study, and cell mediated immune responses which have made them crayfish were protected fully from WSSV following injection attractive for the aquaculture industry (Heppel and Davis with fusion VP19+VP28 polyclonal antiserum (Li et al. 2000). DNA vaccination has already been proven to be effec 2005). Vaccination trials with VP292, a newly identified tive in rainbow trout for infectious haematopoietic necrosis envelope protein, also resulted in significant resistance to virus (Boudinot et al. 1998; Corbeil et al. 1999: Kim et al. WSSV for up to 30 days post initial vaccination (Vaseeharan 2000; Kurathetal. 2006; Lorenzenet al. 2001; Lorenzenet al. et al. 2006). Using a different strategy, Robalino et al. (2004, 1999) and viral haemorrhagic septicemia virus (Lorenzenet 2005) and Tirosophon et al. (2005) demonstrated that the al. 2002) as well as channel catfish for herpes virus 1 (Nus administration of dsRNA specific for WSSV genes induces a US 2008/0107652 A1 May 8, 2008

potent and virus-specific antiviral response in shrimp. Both unicellular algae, multicellular algae, or a combination studies revealed significant reduction in mortality in the thereof. In one embodiment, the microorganism is a cynobac shrimp population protected by Vp28 and vp19 dsRNA injec teria, Dunaliella or a variant thereof. tions. 0033. In the present invention, the microorganism is trans 0026. These approaches to controlling WSSV involve formed with a genetic material the expression products of induction of an immune response to virulence epitopes of which is one or more recombinant molecules comprising one WSSV and suggest that this could potentially control this or more antiviral or antibacterial molecules, immunogenic disease. In each approach, however, vaccine delivery con peptides, single chain antibody fragments, or a combination strains implementation. The method used in the studies cited thereof. The recombinant molecules also comprise cecropins, above, individual inoculation of shrimp, is highly impractical penaeidins, bactenecins, calinectins, myticins, tachyplesins, under field conditions. Given that a typical shrimp grow-out clavanins, misgurins, pleurocidins, parasins, histones, acid pond can harbor upwards of 300,000 post-larvae per hectare, proteins, and lysozymes, or a combination thereof, among labor costs imposed by this method rule out commercial others. application. The coating of dry feed with inoculum appears logical, but the feeding behavior of shrimp involves the slow 0034. The single chain antibody fragment comprises schv. nibbling of feed particles. This behavior will cause substan The single chain antibody fragment blocks assembly of the tial losses of inoculum through leaching. It has been demon virus or bacteria by inhibiting expression of one or more viral strated that within an hour, shrimp feed can lose more than or bacterial proteins. 20% of its crude protein, about 50% of its carbohydrates and 0035. The antibacterial molecule includes, inter alia, 85 to 95% of its vitamin content (Rosenberry 2005). In light Peneidin-Like antimicrobial peptide AMP, among other anti of the tremendous global impact of WSSV on shrimp farming bacterial molecules. and the necessity of high-intensity cultivation, new strategies to impart immunity against WSSV are essential. It is also 0036. The pathogens include virus, bacterium, protozoa, critical that Such a technology be economically viable, Scal or poisons derived from algae, or a combination thereof. In able to large shrimp farming facilities, and easily delivered to one embodiment, the pathogens include Vibrio harveyi, the shrimp. White Spot Syndrome Virus, Taura, variants or serotypes 0027 U.S. Patent application No. 20030211089 discloses thereof. delivery systems and methods for delivering a biologically 0037. In another embodiment, the recombinant molecule active protein to a host animal. The systems and methods is a DNA vaccine. The DNA vaccine can be codon optimized provided include obtaining an algal cell transformed by an for expression in a specific microorganism and/or the target expression vector. The biologically active protein is an anti aquatic animal. gen that upon administration to the animal induces a general 0038. In yet another embodiment, the genetically modi immune response in the host animal. fied microorganism is bioamplified in a probiotic organism 0028 U.S. Patent application No. 20040081638 discloses prior to consumption by the aquatic animal. delivery of disease control in aquaculture and agriculture using nutritional feeds containing bioactive proteins pro 0039 The probiotic organism comprises bacterium, and duced by viruses. The gene encoding a protein orantibody is planktonic organisms comprising Artemia, rotifers, copep incorporated into a virus, which in turn, infects an insect ods, or daphnia, or a combination thereof. organism that is a component of the feed. The virus can infect 0040. In one embodiment, the microorganism is a micro the macroalgal, plant, or animal feed component. algae and transformation of micro algae is achieved by the 0029. The invention, as disclosed and described herein, genetic transformation of a symbiotic or commensal bacteria overcomes the prior art problems by providing novel of the micro algae with a genetic material that expresses in approaches of paratransgenesis for transferring immuno Vivo immunogenic peptides or antibody molecules against genic peptides and antibody fragments that targets specifi pathogenic infections of aquatic animal. cally one or more key epitopes of a pathogen that infects an 0041. In one embodiment, the microalgae comprises Iso aquatic animal. chrysis, Pavlova, Nannochloropsis, Thalassiosira psue donana, Cyanobacterium, Dunaliella, Phaeodactylum tri III. SUMMARY OF THE INVENTION cornutum, Red alga Porphydium cruentum, Haematococcus, 0030 The invention provides methods of paratransgenesis Botryococcus, Gymnodinium sp., Gonyaulax, Chlamydomo for the prevention, amelioration or treatment of a disease or nas, Chlorella pyrenoidosa, or species and variants thereof. disorder in an aquatic animal comprising: i) providing a The cyanobacterium comprises Cyanobacterium Spirulina, genetically modified microorganism that expresses one or cyanobacteria Scytoinema, cyanobacteria Oscillatoria, or more recombinant molecules that specifically target one or Synechococcus bacillarus, or species and variants thereof. more key epitopes of apathogen that infects an aquatic animal 0042. In one embodiment, the aquatic animal is a farm and ii) feeding the aquatic animal directly or indirectly with raised or wild animal. In a preferred embodiment the aquatic the genetically modified microalgae. animal is a farm-raised shrimp. 0031. In one embodiment, the pathogen specifically 0043. In another embodiment, the genetically modified infects the aquatic animal. micro algae expresses a protein, a peptide, or one or more 0032. In another embodiment, the microorganism com antibody fragments that inhibit the growth or replication of a prises algae, bacteria, or a combination thereof. In one shrimp pathogen comprising Vibrio species, Taura, and White embodiment, the microorganism is a microalga, macro alga, spot virus. US 2008/0107652 A1 May 8, 2008

0044. In yet another embodiment, the invention as 0052 The term microorganism has been used to include described herein specifically excludes by way of proviso microalgae herein. those methods for the prevention, amelioration or treatment 0053 As used herein, the term “cyanobacteria' refers to of diseases or disorders in aquatic animals that use genetic prokaryotic organisms formerly classified as the blue-green transformation methods to generally boost the immune algae. Cyanobacteria are a large and diverse group of photo response of an aquatic animal to non-specific pathogens. synthetic bacteria which comprise the largest Subgroup of 0045. This invention as disclosed and described herein Gram-negative bacteria. Cyanobacteria were classified as also expressly excludes the use of transformed insects or algae for many years due to their ability to perform oxygen larvae thereof in delivering the genetic material or the recom evolving photosynthesis. While many cyanobacteria have a binant molecules of the invention to the aquatic animal or to mucilaginous sheath which exhibits a characteristic blue any intermediate hosts and/or feed organisms, including pro green color, the sheaths in different species may also exhibit biotic organisms, that are within the feeding cascade of the colors including light gold, yellow, brown, red, emerald paratransgenesis methods of the invention. green, blue, violet, and blue-black. Cyanobacteria include 0046. These and other aspects and embodiments of the Microcystis aeruginosa, Trichodesmium erythraeum, Apha invention are disclosed in detail herein. nizomenon flos-aquae, and Anabaena flos-aquae. 0054 As used herein, the term “probiotic organisms' IV. BRIEF DESCRIPTION OF THE FIGURES refers to organisms that act assist in amplification of the 0047 FIG. 1: Schematic demonstrating the process of bio genetic material before being consumed by the target aquatic amplification. In this strategy, transgenic Dunaliella is ini animal. Probiotic organisms include algae, bacteria, and tially consumed by feed organisms such as Artemia. The fungi. Such as yeast. engorged Artemia is then fed to the target animal. In this 0055 As used herein, the term “gene' or “genetic mate manner, the Supplement is bioamplified as it progresses up the rial” refers to an element or combination of elements that are food chain. capable of being expressed in a cell, either alone or in com bination with other elements. In general, a gene comprises 0.048 FIG. 2: A framework for a paratransgenic approach (from the 5' to the 3' end): (1) a promoter region, which to control shrimp diseases. The cDNA encoding an anti includes a 5' nontranslated leader sequence capable of func pathogen molecule is cloned into a shuttle vector (12), and tioning in prokaryotic and/or eukaryotic cells; (2) a structural expressed in E. coli. Plasmids carrying the recombinant DNA gene or polynucleotide sequence, which codes for the desired (3) is purified and Subsequently used for transforming D. protein; and (3) a 3' nontranslated region, which typically salina or another feed organism (4). The transgenic feed causes the termination of transcription and the polyadenyla organisms is then fed to Artemia larvae (5). Artemia engorged tion of the 3' region of the RNA sequence. Each of these with transgenic feed organisms (6) is then be used to feed elements is operably linked by sequential attachment to the shrimp larvae (7). Production of the anti-pathogen molecule adjacent element. A gene comprising the above elements is within the gut of the shrimp protects the shrimp from targeted inserted by standard recombinant DNA methods into a micro bacteria or viruses, resulting in healthy shrimp that is ready organism. for harvest (8). 0056. As used herein, “promoter” refers to a region of a V. DETAILED DESCRIPTION OF THE DNA sequence active in the initiation and regulation of the INVENTION expression of a structural gene. This sequence of DNA, usu 0049. The invention as described and disclosed herein ally upstream to the coding sequence of a structural gene, uses methods of paratransgenesis in order to control disease controls the expression of the coding region by providing the and disorders of target farmed and wild aquatic animals, to recognition for RNA polymerase and/or other elements maintain equilibrium in the growth environment of these required for transcription to start at the correct site. animals, and to efficiently transfer desirable genes and gene 0057. As used herein, “protein’ is used interchangeably products to the target aquatic animals and their biological with polypeptide, peptide and peptide fragments. flora. 0.058 As used herein, “polynucleotide' includes cDNA, RNA, DNA/RNA hybrid, anti-sense RNA, ribozyme, DEFINITIONS genomic DNA, synthetic forms, and mixed polymers, both 0050. The definitions used in this application are for illus sense and antisense Strands, and may be chemically or bio trative purposes and do not limit the scope of the invention. chemically modified to contain non-natural or derivatized, synthetic, or semi-synthetic nucleotide bases. Also, included 0051. As used herein, the term “microalgae' include both within the scope of the invention are alterations of a wild type prokaryotic and eukaryotic algae that are classed in many or synthetic gene, including but not limited to deletion, inser different genera. Prokaryotic algae are typically referred to as tion, Substitution of one or more nucleotides, or fusion to cyanobacteria or blue-green algae. Eukaryotic micro algae other polynucleotide sequences, provided that such changes come from many different genera, Some of which overlap in the primary sequence of the gene do not alter the expressed with the macro algae, but can be generally differentiated by peptide ability to elicit protective immunity. their size and lack of defined organs. Micro algae can have specialized cell types. Examples of different groups contain 0059. As used herein, “recombinant molecule' includes ing micro algae include, but are not limited to, the Chloro any gene product that is produced in the course of the tran phyta (e.g. Dunaliella), Phodophyta, Phaeophyta, Dino Scription, reverse-transcription, polymerization, translation, phyta, Euglenophyta, Cyanophyta, Prochlorophyta, and post-translation and/or expression of a gene. Recombinant Cryptophyta. molecules include, but are not limited to, proteins, polypep US 2008/0107652 A1 May 8, 2008

tides, peptides, peptide fragments, immunogenic peptides, not affect its symbiotic functions in the host, or the ability of fusion proteins, antibody fragments, polynucleotide mol the host to consume the microorganism, the host that con ecules, DNA vaccine, among others. Sumes or harbors the transgenic microorganism must main tain its growth and reproductive rates when compared to wild 0060. As used herein, “vaccine” refers to compositions type controls, the products expressed by the transgenic micro that result in both active and passive immunizations. Both organism should target the pathogens within the host, genetic pofynucleotides and their expressed gene products are modification of the microorganism should not render them referred as vaccines herein. virulent either to the host or other organisms in the environ 0061 As used herein, “polypeptides’ include any peptide ment, the microorganism chosen to be transformed should not or protein comprising two or more amino acids joined to each be pathogenic to the host, strategies for foreign gene dispersal other by peptide bonds. As used herein, the term refers to both should target the host and selectively minimize non-target short chains, which also commonly are referred to in the art as uptake and retention of the genetic material. peptides, oligopeptides and oligomers, for example, and to 0066 Probiotics are defined as micro-organisms that are longer chains, which generally are referred to in the art as beneficial to the health of the host. They are not therapeutic proteins, of which there are many types. “Polypeptides’ agents but, instead, directly or indirectly alter the composi include, for example, biologically active fragments, Substan tion of the microbial community in the rearing environment tially homologous polypeptides, oligopeptide, homodimers, or in the gut of the host. Although the mode of action of heterodimers, variants of the polypeptides, modified probiotics is not fully understood, it is likely that they func polypeptides, derivatives, analogs, fusion proteins, agonists, tion by competitive elusion, that is, they antagonize the poten antagonists, or antibody of the polypeptide, among others. tial pathogen by the production of inhibitory compounds or The polypeptides include natural peptides, recombinant pep by competition for nutrients and/or space. It is also likely that tides, synthetic peptides, or a combination thereof. probiotics stimulate a humoral and/or cellular response in the 0062. As used herein, the term “antibody fragments' host. refers to immunogenic or antigenic binding immunoglobulin 0067 Probiotics are usually introduced as part of the feed peptides which are at least about 5 to about 15 amino acids or ing regimen or applied directly to the water. A variety of more in length, and which retain some biological activity or micro-organisms, ranging from aerobic Gram-positive bac immunological activity of an immunoglobulin. The invention teria (e.g., Bacillus spp), to Gram-negative bacteria (Vibro provides a novel approach to control of infectious diseases of spp) and yeast have been utilized successfully to increase the commercial mariculture. In particular, the invention provides commercial yield of farmed marine animals. Several species a method of delivering therapeutic molecules to an aquatic of micro algae have also effectively been used for this pur animal by methods of paratransgenesis that involves admin pose. Of note, the unicellular algae, Tetraselmis suecica, has istration of a feed comprising transgenic micro algae, been used as feed for penaeids and salmonids with significant expressing a recombinant molecule that targets one or more reduction in the level of bacterial diseases. The antagonism key epitopes of a pathogen specific to the aquatic animal. among microorganisms is a naturally occurring phenomenon Paratransgenesis employs genetically transformed microor through which pathogens can be killed or reduced in number ganisms that are in Symbiotic relationship with an intermedi in the aquaculture environment. In aquaculture, where micro ate host, or the target aquatic animal host. The microorgan algae are used as the main live food, the Survival rates of isms are closely linked to these hosts act as a Trojan Horse' prawns, crabs and finfish are not considered to be sufficiently to deliver neutralizing peptides and antibody fragments to the high. However, if certain species of bacteria are present with site of pathogen transmission within the host. An application the algae, the Survival rate increases significantly. It is there of this method involves the expression of peptides and anti fore preferable to feed microorganisms to fish along with body fragments that specifically target key epitopes of patho algae, although the control of these microorganisms is essen gens of commercial mariculture and other types of aquacul tial to prevent the pathogens from dominating the microbial ture. communities. These results have led to further studies using 0063. The transfer of the genetically modified microor viruses, fungi and protozoa as biocontrol agents to eliminate ganism to the target animal occurs through a natural biologi pathogenic organisms. The paratransgenesis method of the cal process such as, for example, feeding the target animal invention complements biocontrol strategies at a molecular with the transgenic microorganism directly or via bioampli level in preventing or treating infectious diseas of aquaculture fication through probiotics. while maintaining the natural balance in their habitat, helping to maintain Suitable environmental conditions in aquaculture 0064. Lines of marine cyanobacteria, algae and diatoms and promoting the growth and health of aquaculture in a most that are common components offeed for farmed shrimp and efficient and environmentally friendly manner. fish have been transformed to produce antibodies that neu tralize infectious pathogens such as WSSV and Vibrio. Deliv 0068 Paratransgenic methods of the invention demon ery of these feed organisms, either in slurry preparations or strate environmentally acceptable approaches for control of via a bioamplification strategy with a probiotic organism Such marine effective control of infections in mariculture and offer robust and pathogen transmission. The risk assessment as, for example, Artemia, resulted in passive immunization of framework being developed for paratransgenic control of the alimentary tract of farmed marine animals. arthropod-borne diseases can be applied in part to maricul 0065. The microorganism used for a paratransgenic ture. Unique aspects of the marine environment, Such as novel approach should satisfy the following requirements. The microflora and fauna, physical and chemical features of microorganism should be amenable to genetic manipulation, marine ecosystems and complex interactions through marine transformation of the microorganism should not alter their food chains were modeled and evaluated carefully during fitness, genetic manipulation of the microorganism should development of paratransgenic interventions. US 2008/0107652 A1 May 8, 2008

0069 Pathogens within the scope of the invention include 0073. According to one embodiment of the invention a wider variety of agents that specifically infect mariculture. described herein, there are provided expression vectors con Pathogens include viral or bacterial pathogens as well as taining one or more gene constructs of the invention carrying toxins produced by algae Such as, for example, dinoflagel the antibody genes, including antibody Subunit genes or frag lates. These pathogens include, by way of example and not ments thereof. The expression vectors of the invention con limitations, White Spot Syndrome Virus (WSSV), species of tain the necessary elements to accomplish genetic transfor Vibrio (including V anguillarum and V ordalii, Vibrio salmo mation of microorganisms so that the gene constructs are nicida, Vibrio harveyi), causative agents and virus for infec introduced into the microorganism's genetic material in a tious hypodermal and haematopoietic necrosis (IHHN) and stable manner, i.e., a manner that will allow the antibody IHHNV. causative agent for run-deformity syndrome or RDS genes to be passed on the microorganism’s progeny. The of Penaeus vannamei, Baculo-like viruses, Infectious Pancre design and construction of the expression vectors influence atic Necrosis Virus (IPNV), Hirame rhabdovirus (HIRRV), the integration of the gene constructs into the microorganism the Yellowtail Ascites Virus (YAV), Striped Jack Nervous genome and the ability of the antibody genes to be expressed Necrosis Virus (SJNNV), Irido, Aeromonos hydrophila, by microorganism cells. Aeromonos Salmonicida, Serratia liquefaciens, Yersnia ruck 0074 Preferred among expression vectors are vectors car eri type I, Infectious salmon anaemia (USA) virus, Pancreas rying a functionally complete human or mammalian heavy or Disease (PD), Viral Hemorrhagic Septicemia (VHS), Renni light chain sequence having appropriate restriction sites engi bacterium salmoninarum, Aeromonas salmonicida, Aeromo neered so that any variable V or variable V chain sequence nas hydrophila, species of Pasteurella (including P. pisci with appropriate cohesive ends can be easily inserted therein. cida), species of Yersinia, species of Streptococcus, Human C or C chain sequence-containing vectors are thus Edwardsiella tarda and Edwardsiella ictaluria; the viruses an embodiment of the invention and can be used as interme causing viral hemorrhagic septicemia, infectious pancreatic diates for the expression of any desired complete Hor L chain necrosis, Viremia of carp, channel catfish virus, grass carp in any appropriate host. hemorrhagic virus, nodaviridae Such as nervous necrosis virus, infectious salmon anaemia virus; and the parasites Cer 0075. Many vector systems are available for the expres atomyxa Shasta, Ichthyophthirius multifillius, Cryptobia sion of cloned HC and LC genes in host cells. Different salmositica, Lepeophtherius salmonis, Tetrahymena species, approaches can be followed to obtain complete HC and LC Trichodina species and Epistylus species, dinoflagellates tox subunit antibodies. In one embodiment, HC and LC were ins including toxins causing Diaarhetic Shellfish Poisoning co-expressed in the same cells to achieve intracellular asso (DSP), Paralytic Shellfish Poisoning (PSP), Neurotoxin poi ciation and linkage of HC and LC into complete tetrameric soning (NSP) and Ciguatera, and many more, all of which HC and LC antibodies. The co-expression can occur by using cause serious damage in aquaculture. either the same or different plasmids in the same host. 0.076 Polynucleotides encoding both HC and LC are 0070. In a preferred embodiment, the method of the inven placed under the control of one or more different or the same tion employs genetically transformed cyanobacteria or promoters, for example in the form of a dicistronic operon, Dunaliella that express P monodon antiviral protein (PmAV) into the same or different expression vectors. The expression and P. monodon antimicrobial peptide (PmAMP). vectors are then transformed into cells, thereby selecting 0071 Aquatic animals includes vertebrates, invertebrates, directly for cells that express both chains. arthropods, fish, mollusks, including, by way of example and 0077. In one embodiment, the polynucleotide encoding not limitation, shrimp (e.g., penaeid shrimp, brine shrimp, LC and polynucleotides encoding HC are present on two freshwater shrimp, etc), crabs, oysters, Scallop, prawn clams, mutually compatible expression vectors which are each under cartilaginous fish (e.g., bass, striped bass, tilapia, catfish, Sea the control of different or the same promoter(s). In this bream, rainbow trout, Zebrafish, red drum, salmonids, carp, embodiment, the expression vectors are co-transformed or catfish, yellowtail, carp, etc), crustaceans, among others. transformed individually. For example, cells are transformed Shrimp includes all variety and species of shrimp, including first with an expression vector encoding one chain, for by way of example and not limitation, Penaeus stylirostris, example LC, followed by transformation of the resulting cell Penaeus vannamei, Penaeus monodon, Penaeus chinensis, with an expression vector encoding a iiC. Penaeus Occidentalis, Penaeus Californiensis, Penaeus 0078. In another embodiment, a single expression vector semisulcatus, Penaeus monodon, Penaeus esculentu, carrying polynucleotides encoding both the HC and LC is Penaeus setiferus, Penaeus japonicus, Penaeus aztecus, used. Cell lines expressing HC and LC molecules could be Penaeus duorarum, Penaeus indicus, and Penaeus merguien transformed with expression vectors encoding additional sis, among others species of shrimp. copies of LC, HC, or LC plus HC in conjunction with addi Expression Vectors tional selectable markers to generate cell lines with enhanced 0072 Also encompassed within the scope of the invention properties, such as higher production of assembled HC and are expression vectors containing the gene constructs of the LC antibody molecules or enhanced stability of the trans invention. Expression vectors are defined herein as DNA formed cell lines. sequences that are required for the transcription of cloned 0079 Specifically designed expression vectors allow the copies of genes and the translation of their mRNAS in an shuttling of DNA between hosts, such as between bacteria appropriate host. Such expression vectors are used to express plant or bacteria-animal cells. According to a preferred eukaryotic and prokaryotic genes in a variety of hosts such as embodiment of the invention, the expression vector contains bacteria, yeast, plant cells, fungi, insect cells and animal cells. an origin of replication for autonomous replication in host Expression vectors include, but are not limited to, cloning cells, selectable markers, a limited number of useful restric vectors, modified cloning vectors, specifically, designed plas tion enzyme sites, active promoter(s), and additional regula mids or viruses. tory control sequences. US 2008/0107652 A1 May 8, 2008

0080 Preferred among expression vectors, in certain 0089 Examples of an inducible and/or constitutive pro embodiments, are those expression vectors that contain cis moters include, but are not limited to, promoters isolated from acting control regions effective for expression in a host opera the caulimovirus group Such as the cauliflower mosaic virus tively linked to the polynucleotide of the invention to be 35S promoter (CaMV35S), the enhanced cauliflower mosaic expressed. Appropriate trans-acting factors are Supplied by virus 35S promoter (enh CaMV35S), the figwort mosaic virus the host, Supplied by a complementing vector or Supplied by full-length transcript promoter (FMV35S), the promoter iso the vector itself upon introduction into the host. lated from the chlorophyll a?b binding protein, proteinase 0081. In certain preferred embodiments in this regard, the inhibitors (PI-I, PI-II), defense response genes, phytoalexin expression vectors provide for specific expression. Such spe biosynthesis, phenylpropanoid phytoalexin, phenylalanine cific expression is an inducible expression, cell or organ spe ammonia lyase (PAL), 4-coumarate CoA ligase (4CL), chal cone synthase (CHS), chalcone isomerase (CHI), resveratrol cific expression, host-specific expression, or a combination (stilbene) synthase, isoflavone reductase (IFR), terpenoid thereof. phytoalexins, HMG-CoA reductase (HMG), casbene syn 0082 Promoters thetase, cell wall components, lignin, phenylalanine ammo 0083 Promoters are responsible for the regulation of the nia lyase, cinnamyl alcohol dehydrogenase (CAD), caffeic transcription of DNA into mRNA. A number of promoters acid o-methyltransferase, lignin-forming peroxidase, which function in microorganism cells are known in the art, hydroxyproline-rich glycoproteins (HRGP), glycine-rich and may be employed in the practice of the present invention. proteins (GRP), thionins, hydrolases, lytic enzymes, chiti These promoters are obtained from a variety of sources such nases (PR-P, PR-Q), class I chitinase, basic, Class I and II as, for example, viruses, plant, and bacteria, among others. chitinase, acidic, class II chitinase, bifunctional lysozyme, B-1,3-Glucanase, arabidopsis, B-fructosidase, Superoxide 0084. The invention, as described and disclosed herein, dismutase (SOD), lipoxygenase, prot., PR1 family, PR2, encompasses the use of constitutive promoters, inducible pro PR3, osmotin, PR5, ubiquitin, wound-inducible genes, win1. moters, or both. In general, an “inducible promoter is a win2 (hevein-like), Wunl, wun2, nos, nopaline synthase, promoter that is capable of directly or indirectly activating ACC synthase, HMG-CoA reductasehmg1,3-deoxy-D-ara transcription of one or more DNA sequences or genes in bino-heptulosonate-7-phosphate synthase, HSP7033, Sali response to an inducer. In the absence of an inducer the DNA cylic acid inducible, acid peroxidase, PR-proteins, glycine sequences or genes will not be transcribed. Typically the rich protein, methyljasmonate inducible, vspE", heat-shock protein factor that binds specifically to an inducible promoter genes, HSP70, cold-stress inducible, drought, salt stress, hor to activate transcription is present in an inactive form which is mone inducible, gibberellin, O-amylase, abscisic acid, EM-1, then directly or indirectly converted to the active form by the RAB, LEA genes, ethylene, phytoalexin biosyn genes, or a inducer. The inducer can be a chemical agent Such as a pro combination thereof among others. tein, metabolite, growth regulator, herbicide orphenolic com pound or a physiological stress imposed directly by heat, 0090 The above-noted promoters are listed solely by way cold, wound, salt, or toxic elements, light, desiccation, patho of illustration of the many commercially available and well gen infection, or pest-infestation. known promoters that are available to those of skill in the art for use inaccordance with this aspect of the present invention. 0085 Inducible promoters are determined using any It will be appreciated that any other promoter suitable for, for methods known in the art. For example, the promoter may be example, introduction, maintenance, propagation or expres operably associated with an assayable marker gene Such as sion of a polynucleotide or polypeptide of the invention in GUS (glucouronidase), the host microorganism can be engi microorganism may be used in this aspect of the invention. neered with the construct; and the ability and activity of the promoter to drive the expression of the marker gene in the 0.091 Regulatory Control Elements harvested tissue under various conditions assayed. 0092 Gene constructs or genetic material of the present 0.086 A microorganism cell containing an inducible pro invention can also include other optional regulatory elements moter is exposed to an inducer by externally applying the that regulate, as well as engender, expression. Generally Such inducer to the cell or microorganism such as by spraying, regulatory control elements-operate by controlling transcrip harvesting, watering, heating or similar methods. A "consti tion. Examples of Such regulatory control elements include, tutive promoter is a promoter that directs the expression of a for example, enhancers (either translational or transcriptional gene throughout the various parts of an organism and con enhancers as may be required), repressor binding sites, ter tinuously throughout development of the organism. minators, leader sequences, and the like. 0087. In one embodiment of the invention, promoters are 0093 Specific examples of these elements include, the tissue-specific. Non-tissue-specific promoters (i.e., those that enhancer region of the 35S regulatory region, as well as other express in all tissues after induction), however, are preferred. enhancers obtained from other regulatory regions, and/or the More preferred are promoters that additionally have no or ATG initiation codon and adjacent sequences. The initiation very low activity in the uninduced state. Most preferred are codon must be in phase with the reading frame of the coding promoters that additionally have very high activity after sequence to ensure translation of the entire sequence. The induction. Particularly preferred among inducible promoters translation control signals and initiation codons are from a are those that can be induced to express a protein by environ variety of origins, both natural and synthetic. Translational mental factors that are easy to manipulate. initiation regions are provided from the Source of the tran 0088. In a preferred embodiment of the invention, one or Scriptional initiation region, or from the structural gene. The more constitutive promoters are used to regulate expression sequence is also derived from the promoter selected to of the antibody genes or antibody Subunit genes in microor express the gene, and can be specifically modified to increase ganisms. translation of the mRNA. US 2008/0107652 A1 May 8, 2008

0094. The nontranslated leader sequence is derived from These methods involve strategies utilizing, for example, any suitable source and is specifically modified to increase the direct terminal repeats, inverted terminal repeats, double translation of the mRNA. In one embodiment, the 5' nontrans expression cassette knock-in, specific gene knock-in, specific lated region is obtained from the promoter selected to express gene knock-out, random chemical mutagenesis, random the gene, the native leader sequence of the gene, coding mutagenesis via transposon, and the like. The expression region to be expressed, viral RNAS, Suitable eucaryotic genes, vectoris, for example, flanked with homologous sequences of or a synthetic gene sequence, among others. any non-essential microorganism genes, transposon 0.095. In another embodiment, gene constructs of the sequence, or ribosomal genes. The DNA is then integrated in present invention comprise a 3U untranslated region. A 3U host by homologous recombination occurred in the flanking untranslated region refers to that portion of a gene comprising sequences using standard techniques. a DNA segment that contains a polyadenylation signal and 0102. In other embodiments, various alternative methods any other regulatory signals capable of effecting mRNA pro for introducing recombinant nucleic acid constructs into cessing or gene expression. The polyadenylation signal is microorganisms are also utilized. Alternative gene transfer usually characterized by effecting the addition of polyade and transformation methods include, but are not limited to, nylic acid tracks to the 3U end of the mRNA precursor. electroporation-mediated uptake of naked DNA, microinjec 0096. The termination region or 3' nontranslated region is tion, silicon carbide mediated DNA uptake, and microprojec employed to cause the termination of transcription and the tile bombardment, among others. addition of polyadenylated ribonucleotides to the 3' end of the 0.103 In the case of direct genetransfer, the gene construct transcribed mRNA sequence. The termination region may be is transformed into microorganism without the use of plas native with the promoter region, native with the structural mids. Direct transformation involves the uptake of exogenous gene, or may be derived from the expression vector or another genetic material into microorganism. Such uptake may be Source, and would preferably include a terminator and a enhanced by use of chemical agents or electric fields. The sequence coding for polyadenylation. The addition of appro exogenous material may then be integrated into the nuclear priate introns and/or modifications of coding sequences for genome. Alternatively, exogenous DNA can be introduced increased translation can also Substantially improve foreign into cells or by microinjection. In this technique, a Solution of gene expression. the plasmid DNA or DNA fragment is injected directly into 0097 Selectable Markers the cell with a finely pulled glass needle. A more recently developed procedure for direct gene transfer involves bom 0.098 To aid in identification of transformed microorgan bardment of cells by micro-projectiles carrying DNA. In this ism cells, the gene constructs of this invention may be further procedure, commonly called particle bombardment, tungsten manipulated to include selectable marker genes that are func or gold particles coated with the exogenous DNA are accel tional in bacteria, algae, and/oraquatic host. Useful selectable erated toward the target cells. The particles penetrate the cells markers include, but are not limited to, enzymes which pro carrying with them the coated DNA. Microparticle accelera vide for resistance to an antibiotic Such as Ampicillin resis tion has been successfully demonstrated to lead to both tran tance gene (Amp'), tetracycline resistance gene (Tcr), Cyclo sient expression and stable expression in cells Suspended in heximide-resistance L41 gene, the gene conferring resistance cultures. to Antibiotic G418 such as the APT gene derived from a bacterial transposon Tn)03, the antibiotic Hygromycin B-re 0104. Use of Vaccines in Mariculture sistance gene, Gentamycin resistance gene, and/or kanamy 0105 The use of antibodies for therapeutic and diagnostic cine resistance gene, among others. Similarly, enzymes pro purposes has gained prominence in the past decade. Immu viding for production of a compound identifiable by color noglobulins are very specific to their targets and could be used change such as GUS, or luminescence, such as luciferase are to design high affinity-based reagents for immunotherapeutic included herein. applications. Problems associated with the relatively short 0099. A selectable marker gene is used to select transgenic half life of passively administered immunoglobulins can be microorganism cells of the invention, which transgenic cells overcome by using constitutively-expressed single chain have integrated therein one or more copies of the gene con antibodies (scFv), instead of whole IgG molecules. These are struct of the invention. The selectable or screenable genes Smaller in size and can be synthesized as bivalent to multiva provide another control for the successful culturing of cells lent molecules that can attack different targets on the patho carrying the genes of interest. Transformed microorganism gen. may be selected by growing the cells on a medium containing, 0106 The invention provides for genetic materials that for example, Kanamycin. encode antibody fragments that are expressed within the 0100 Transformation Strategies microorganism before consumption by the aquatic animal. In one embodiment, the antibodies include immunoglobulin 0101 Microorganisms are genetically transformed to molecules having H and L chains associated therein so that incorporate one or more gene constructs of the invention. the overall molecule exhibits the desired binding and recog There are numerous factors which influence the success of nition properties. Various types of immunoglobulin mol transformation. The design and construction of the expres sion vector influence the integration of the foreign genes into ecules are provided: monovalent, divalent, or molecules with the genome of the microorganism and the ability of the for the specificity-determining V binding domains attached to eign genes to be expressed by the microorganism. The inte moieties carrying desired functions. gration of the polynucleotides encoding the desired gene into 0.107 In another embodiment, the invention provides for the microorganism is achieved through strategies that genetic material encoding fragments of chimeric immunoglo involve, for example, insertion or replacement methods. bulin molecules such as Fab, Fab', or F(ab') molecules or US 2008/0107652 A1 May 8, 2008

those proteins coded by truncated genes to yield molecular naturally as allelic variations, as disclosed above, due to species functionally resembling these fragments. A chimeric genetic polymorphism, for example, or may be produced by chain contains a constant (C) region Substantially similar to human intervention (i.e., by mutagenesis of cloned DNA that present in a natural mammalian immunoglobulin, and a sequences). Such as induced point, deletion, insertion and variable (V) region having the desired anti-pathogenic speci Substitution mutants. Minor changes in amino acid sequence ficity of the invention. Antibodies having chimeric H chains are generally preferred. Such as conservative amino acid and L chains of the same or different V region binding speci replacements, Small internal deletions or insertions, and addi ficity are prepared by appropriate association of the desired tions or deletions at the ends of the molecules. polypeptide chains. 0113. It has been demonstrated that human monoclonal 0108. The immunoglobulin molecules are encoded by antibodies can be expressed in transgenic algae chloroplasts. genes which include the kappa, lambda, alpha, gamma, delta, C. reinhardtii chloroplast atpA or rbcL promoters and its 5' epsilon and mu constant regions, as well as any number of untranslated regions were used to drive expression of an immunoglobulin variable regions. Light chains are classified engineered large single-chain antibody gene in this algae. as either kappa or lambda. Light chains comprise a variable This antibody is directed against herpes simplex virus (HSV) light (V) and a constant light (C) domain. Heavy chains are glycoprotein D and accumulates as a functional Soluble pro classified as gamma, mu, alpha, delta, or epsilon, which in tein in transgenic chloroplasts, and binds herpes virus pro turn define the immunoglobulin classes IgG, IgM, IgA, Ig) teins, as determined by ELISA assays. These studies demon and IgE, respectively. Heavy chains comprise variable heavy strated that algae can be used as an expression platform to (V), constant heavy 1 (CH), hinge, constant heavy 2 (CH), synthesize complex recombinant proteins. and constant heavy 3 (CH) domains. The mammalian IgG heavy chains are further sub-classified based on their 0114 Costs for production of recombinant proteins in sequence variation, and the Subclasses are designated IgG1. algal systems are quite reasonable (S0.002 per liter). In addi IgG2, IgG3 and IgG4. tion, algae can be grown in continuous culture, their growth medium can be recycled, transgenic algae can be generated 0109 Antibodies can be further broken down into two quickly, as it requires only a few weeks between the genera pairs of a light and heavy domain. The paired V and V. tion of initial transformants and their scale-up to production domains each comprise a series of seven Subdomains: frame Volumes, and finally, the chloroplast and nuclear genome of work region 1 (FR1), complementarity determining region 1 algae can be genetically transformed opening the possibility (CDR1), framework region 2 (FR2), complementarity deter of producing any transgenic protein in a single organism. mining region 2 (CDR2), framework region 3 (FR3), comple mentarity determining region 3 (CDR3), framework region 4 0115 Chimeric antibody technology bridges both the (FR4) which constitute the antibody-antigen recognition hybridoma and genetic engineering technologies to provide domain, etc. recombinant molecules for the prevention and treatment of infections in marine culture. The chimeric antibodies of the 0110. In general, as used herein, the term antibody or present invention embody a combination of the advantageous antibody fragment of the invention encompasses variety of characteristics of mAbs. Like mouse mabs, they can recog modifications, particularly those that are present in polypep nize and bind to a specific epitope of an antigen present in the tides expressed by polynucleotides in a host cell. It will be target animal. Moreover, using the methods disclosed in the appreciated that polypeptides often contain amino acids other present invention, any desired antibody isotype can be com than the 20 amino acids commonly referred to as the 20 bined with any particular antigen combining site. naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a 0116. In one embodiment, the invention provides cyano given polypeptide, either by natural processes, such as pro bacteria or microalgae-derived mammalian or chimericanti cessing and other post-translational modifications, or by bodies, including antibody subunits and fragments thereof, chemical modification techniques. with specificity to a pathogen of marinculture. 0111 Modifications occur anywhere in a polypeptide, 0117. In another embodiment, Synechococcus bacillarus, including the peptide backbone, the amino acid side chains a cyanobacterium, was transformed with a DNA construct and the amino or carboxyl termini. Blockage of the amino or that encodes a single chain antibody and can stably express carboxyl group in a polypeptide, or both, by a covalent modi the corresponding Schvin its functional state. In this study, an fication, occurs in natural or synthetic polypeptides and Such expression plasmid encoding the murine single chain anti modifications may be present in the antibody polypeptides of body, rDB3 was used (Durvasula et al. 1999, incorporated the present invention, as well. In general, the nature and herein by reference in its entirety). This study confirms that extent of the modifications are determined by the host cells genetically modified cyanobacteria can be used as a delivery post-translational modification capacity and the modification system to secrete anti-pathogen molecules that affect shrimp signals present in the polypeptide amino acid sequence. It will and mollusks, as part of a paratransgenic strategy to control be appreciated that the same type of modification may be infectious diseases of mariculture. present in the same or varying degrees at several sites in a 0118. In another embodiment, immune peptides and anti polypeptide. body fragments were expressed in transgenic Chlorophyta 0112 The microorganism-derived antibody according to spp. In particular, single chain antibody fragments were the invention includes truncated and/or N-terminally or C-ter developed that target key epitopes of marine pathogens Such minally extended forms of the antibody, analogs having as, for example, Vibrio spp., White Spot Syndrome Virus amino acid Substitutions, additions and/or deletions, allelic (WSSV), or a combination thereof, among others. Central to variants and derivatives of the antibody. Variations in the all of our paratransgenic approaches is the concept of co structure of microorganism-derived antibodies may arise expression of multiple immune peptides and antibody frag US 2008/0107652 A1 May 8, 2008

ments, to minimize evolution of resistance amongst target in many regions, identical. As noted below, changes in the pathogens. As delivery systems are refined to disperse engi nucleotide sequence of the variant may be silent. That is, they neered algae and cyanobacteria into populations of shrimp, may not alter the amino acids encoded by the polynucleotide. shellfish and fish, the invention has deployed multiple strains Where alterations are limited to silent changes of this type, a of engineered organisms that target unique pathogen variant will encode a polypeptide with the same amino acid epitopes. sequence as the reference. 0119 Expression of immune peptides and engineered 0.126 Changes in the nucleotide sequence of the variant single chain antibody fragments by organisms that are used as may alter the amino acid sequence of a polypeptide encoded feed in mariculture operations offers a novel strategy to by the reference polynucleotide. Such nucleotide changes deliver passive immunity to the gut of farmed shrimp, shell may result in amino acid Substitutions, additions, deletions, fish and fish. Since many pathogens gain access via the diges fusions and truncations in the polypeptide encoded by the tive tract, this approach enhances the arsenal against several reference sequence. According to a preferred embodiment of infections that currently afflict mariculture operations. the invention, there are no alterations in the amino acid 0120 Polynucleotides Encoding Antibody Polypeptides sequence of the polypeptide encoded by the polynucleotides This invention also encompasses polynucleotides that corre of the invention, as compared with the amino acid sequence of spond to and code for the antibody polypeptides. Nucleic acid the wild type or mammalian derived peptide. sequences are either synthesized using automated systems 0127. The present invention further relates to polynucle well known in the art, or derived from a gene bank. otides that hybridize to the herein described sequences. The 0121. It will be appreciated that a great variety of modifi term "hybridization under stringent conditions' according to cations have been made to DNA and RNA that serve many the present invention is used as described by Sambrook et al. useful purposes known to those of skill in the art. The poly (1989) Molecular Cloning, A Laboratory Manual, Cold nucleotides of the invention embrace chemically, enzymati Spring Harbor Laboratory Press 1.101-1.104. Preferably, a cally or metabolically modified forms of polynucleotides. stringent hybridization according to the present invention is given when after washing for an hour with 1% SSC and 0.1% 0122 The polynucleotides of the present invention SDC at 50° C., preferably at 55° C., more preferably at 62°C., encode, for example, the coding sequence for the structural most preferably at 68°C., a positive hybridization signal is gene (i.e., antibody gene), and additional coding or non still observed. A polynucleotide sequence which hybridizes coding sequences. Examples of additional coding sequences under such washing conditions with the nucleotide sequence include, but are not limited to, sequences encoding a secretory shown in any sequence disclosed herein or with a nucleotide sequence. Such as a pre-, pro-, or prepro-protein sequences. sequence corresponding thereto within the degeneration of Examples of additional non-coding sequences include, but the genetic code is a nucleotide sequence according to the are not limited to, introns and non-coding 5' and 3' sequences, invention. Such as the transcribed, non-translated sequences that play a role in transcription and mRNA processing, including splic 0128. The polynucleotides of the invention include poly ing and polyadenylation signals, for example, for ribosome nucleotide sequences that have at least about 50%. 65%. 70%, binding and stability of mRNA. 75%, 80%, 85%, 90%, 95%, 98%, 99% or more nucleotide sequence identity to the polynucleotides or a transcriptionally 0123 The polynucleotides of the invention also encode a active fragment thereof. To determine the percent identity of polypeptide which is the mature protein plus additional two amino acid sequences or two nucleic acid sequences, the amino or carboxyl-terminal amino acids, oramino acids inte sequences are aligned for optimal comparison purposes (i.e., rior to the mature polypeptide (when the mature form has gaps can be introduced in the sequence of a first amino acid or more than one polypeptide chain, for instance). Such nucleic acid sequence for optimal alignment with a second sequences play a role in, for example, processing of a protein nucleic acid sequence). The amino acid residue or nucleotides from precursor to a mature form, may facilitating protein at corresponding amino acid or nucleotide positions are then trafficking, prolonging or shortening protein half-life or compared. When a position in the first sequence is occupied facilitating manipulation of a protein for assay or production, by the same amino acid residue or nucleotide as the corre among others. The additional amino acids may be processed sponding position in the second sequence, then the molecules away from the mature protein by cellular enzymes. are identical at that position. The percent identity between the 0124. In sum, the polynucleotides of the present invention two sequences is a function of the number of identical posi encode, for example, a mature protein, a mature protein plus tions shared by the sequences (i.e., 9% identity = it of identical a leader sequence (which may be referred to as a preprotein), overlapping positions/total # of positions X 100). In one a precursor of a mature protein having one or more prose embodiment, the two sequences are the same length. quences which are not the leader sequences of a preprotein, or 0129. The determination of percent identity between two a preproprotein, which is a precursor to a proprotein, having sequences also can be accomplished using a mathematical a leader sequence and one or more prosequences, which algorithm. A preferred, non-limiting example of a mathemati generally are removed during processing steps that produce cal algorithm utilized for the comparison of two sequences is active and mature forms of the polypeptide. the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. 0125 The polynucleotides of the invention include “vari Sci. USA 87:2264-2268, modified as in Karlin and Altschul ant(s) of polynucleotides, or polypeptides as the term is used (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an herein. Variants include polynucleotides that differ in nucle algorithm is incorporated into the NBLAST and XBLAST otide sequence from another reference polynucleotide. Gen program of Altschuletal. (1990), J. Mol. Biol. 215:403-410. erally, differences are limited so that the nucleotide sequences BLAST nucleotide searches can be performed with the of the reference and the variant are closely similar overalland, NBLAST program, score=100, wordlength 12 to obtain US 2008/0107652 A1 May 8, 2008 nucleotide sequences homologous to a nucleic acid mol influenza A nucleoprotein (NP) elicited a CD8+ response to ecules of the invention. The BLAST protein searches can be NP that protected mice against challenge with heterologous performed with the XBLAST program, score=50, strains of flu. (Montgomery, et al., Supra). Another advantage wordlength=3 to obtain amino acid sequences homologous of the immunization with a DNA vaccine rather than its gene to a protein molecule of the invention. To obtain gapped product is the relative simplicity with which native or nearly alignments for comparison purposes, Gapped BLAST can be native antigen can be presented to the immune system. Mam utilized as described in Altschul et al (1997) Nucleic Acids malian proteins expressed recombinantly in bacteria, yeast, ReS. 25:3389-34O2. or even mammalian cells often require extensive treatment to 0130. Alternatively, PSI-Blast can be used to perform an ensure appropriate immunogenicity. iterated search which detects distant relationships between 0.137 Also, the ease of producing and purifying DNA molecules (Id.). When utilizing BLAST, Gapped BLAST and constructs compares favorably with traditional protein puri PSI-Blast programs, the default parameters of the respective fication, facilitating the generation of combination vaccines. programs (i.e., XBLAST and NBLAST program can be used. Thus, in accordance with one embodiment of the invention, Another preferred, non-limiting example of a mathematical multiple genes, for example, genes encoding antigens algorithm utilized for the comparison of sequences is the VP19+VP28 of WSSV in combination with other genes algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such encoding any other viral or bacterial binding antigens are an algorithm is incorporated into the ALIGN program (ver transferred to the microalgae according to the methods of the sion 2.0) which is part of the GCG sequence alignment soft invention. ware package. When utilizing the ALIGN program for com paring amino acid sequences of a PAM 120 weight residue 0.138. As is well known in the art, a large number of factors table, a gap length penalty of 12 and a gap penalty of 4 can be can influence the efficiency of expression of antigen genes used. In an alternate embodiment, alignments can be obtained and/or the immunogenicity of DNA vaccines. Examples of using the NA MULTIPLE ALIGNMENT 1.0 program, Such factors include the reproducibility of inoculation, con using a GapWeight of 5 and a Gaplength Weight of 1. struction of the plasmid vector, choice of the promoter used to drive antigen gene expression and stability of the inserted 0131 DNA Vaccines gene in the plasmid. Depending on their origin, promoters 0132. According to one embodiment of the invention, the differ in tissue specificity and efficiency in initiating mRNA recombinant molecule is a DNA vaccine. DNA vaccines, an synthesis (see, for example, Xiang et al., Virology, 209:564 alternative to a traditional Vaccine comprising an antigen and 579 (1994); Chapman et. al., Nucleic Acids. Res., 19:3979 an adjuvant, involve the direct in vivo introduction of DNA 3986 (1991). To date, most DNA vaccines in mammalian encoding the antigen into tissues of an organism for expres systems have relied upon viral promoters derived from sion of the antigen by the cells of the Subject's organism. Such cytomegalovirus (CMV). vaccines are termed herein “DNA vaccines' or “polynucle 0.139. Another factor known to affect the immune otide-based vaccines' DNA vaccines are described in Inter response elicited by DNA immunization is the method of national Patent Publication WO95/20660 and International DNA delivery. For example, high-velocity inoculation of Patent Publication WO 93/19183, the disclosures of which plasmids, using a gene-gun, enhanced the immune responses are hereby incorporated by reference in their entireties. of mice, Eisenbraun et al., DNA Cell Biol., 12: 791-797 0133. In contrast to conventional vaccines, DNA and other (1993), presumably because of a greater efficiency of DNA subunit vaccines exclusively utilize host cell molecules for transfection and more effective antigen presentation by den transcription and translation of proteins. In one embodiment, dritic cells. the DNA vaccine of the invention contains modified codon 0140 Also contemplated within the scope of the invention usage of the host microalgae. is the use of naked polynucleotides, unassociated with any 0134. The ability of directly injected DNA that encodes a plasmids, proteins, adjuvants or other agents which affect the protein to elicit a protective immune response has been dem recipients’ immune system. In this case, it is desirable for the onstrated in numerous experimental systems Conry et al., polynucleotide to be in a physiologically acceptable solution, Cancer Res., 54:1164-1168 (1994); Cox et al., Virol, such as, but not limited to, sterile saline or sterile buffered 67:5664-5667 (1993); Davis et al., Hum. Mole. Genet. saline. Alternatively, the polynucleotides may be associated 2:1847-1851 (1993); Sedegah et al., Proc. Natl. Acad. Sci., with liposomes, such as lecithin liposomes or other liposomes 91:9866-9870 (1994); Montgomery et al., DNA Cell Bio. known in the art, as a DNA-liposome mixture, or associated 12:777-783 (1993); Ulmer et al., Science, 259:1745-1749 with an adjuvant known in the art to boost immune responses, (1993); Wang et al., Proc. Natl. Acad. Sci., 90:4156-4160 Such as a protein or other carrier. (1993); Xiang et al., Virology, 199:132-140 (1994). 0.141 Agents that assist in the cellular uptake of DNA, 0135 Studies with ferrets indicate that DNA vaccines Such as, but not limited to, calcium ions, may also be used. against conserved internal viral proteins of influenza, These agents are generally referred to herein as transfection together with Surface glycoproteins, are more effective facilitating reagents and pharmaceutically acceptable carri against antigenic variants of influenza virus than are either ers. Techniques for coating microprojectiles coated with inactivated or subvirion vaccines Donnelly et al., Nat. Medi polynucleotides are known in the art and are also useful in cine, 6:583-587 (1995). connection with this invention. 0136. One of the advantages of DNA immunization over 0142. This invention is further illustrated by the following antigen immunization is the potential for the immunogen to examples, which are not to be construed in any way as impos enter the MHC class I pathway and evoke a cytotoxic T cell ing limitations upon the scope thereof. On the contrary, it is to response. Immunization of mice with DNA encoding the be clearly understood that resort may be had to various other US 2008/0107652 A1 May 8, 2008

embodiments, modifications, and equivalents thereof which, transgenic cyanobacterium expressed an active recombinant after reading the description herein, may suggest themselves antibody and serves as a model for future applications of this to those skilled in the art without departing from the spirit of technology. the present invention and/or the scope of the appended claims. Example 3 EXAMPLES Bioamplification of Foreign Gene Products Through Example 1 Transgenic Micro Algae 0.148. An alternate strategy for delivery of transgenic Transformation of Dunliella Dunaliella to the target animal is via bioamplification. In this 0143. This example demonstrates the use of Dunaliella for strategy a feed organism Such as Artemia initially consumes paratransgenic control of infectious diseases of farmed the transgenic Dunaliella. The engorged Artemia is then fed shrimp. Paratransgenesis is a strategy that employs commen to the target animal. In this manner, the Supplement is bioam sal or symbiotic organisms to express molecules that interfere plified as it progresses up the food-chain. Artemia are non with transmission cycles of infectious pathogens. Application selective filter feeders and therefore will ingest a wide range of paratransgenesis to viral and bacterial diseases of farmed of foods. The main criteria for food selection are particle size, shrimp has been described with genetically modified cyano digestibility, and nutrient levels. (Dobbeleir et al. 1980). Pos bacteria as the delivery agents. Here, a transformation system sibly the best foods for Artemia are live micro algae such as for Dunaliella and a framework for application of engineered Nannochloropsis, Tetraselmis, Isochrysis, Dunaliella and Dunaliella in commercial mariculture are presented. Pavlova. Combinations of live phytoplanktons fed to Artemia cultures have demonstrated Superior enrichment characteris 0144. D. salina was transformed carrying a construct that tics over feeding single phytoplankton species (D’Agostino contains the chloramphenicol acetyltransferase (CAT) gene 1980). However, not all species of unicellular algae are appro as well as the gene that encodes for green fluorescent protein priate for Sustaining Artemia growth. For example, Chlorella (GFP). Using GFP as a marker, we fed the transformed and Stichococcus have a thick cell wall that cannot be Dunaliella as a slurry to shrimp nauplii and monitor the digested by Artemia. progression of the GFP within the gut. This study demon strated successful delivery of a functional protein from the Example 4 feed to the target organism. 0145 Dunaliella salina is also being transformed to Construction of Shuttle Plasmid pRrMDWK6 and express molecules with activity against infectious pathogens Transformation of Synechococcus bacillarus of mariculture. A gene encoding Penaeidin, an antibacterial 0.149 Electro-competent Synechococcus was generated peptide with activity against Vibrio species, has been synthe by adapting protocols for E. coli. The expression-shuttle plas sized and is incorporated into an expression plasmid for trans mid, pRrMDWK6, was constructed using a gene encoding a formation of Dunaliella. Extracts from transformed lines of murine three-domain VHKantibody fragment (rDB3) which D. Salina have been analyzed for activity against a variety of binds progesterone (He et al., 1995, incorporated herein by gram negative and gram positive bacteria, with special focus reference in its entirety). The expression of this antibody on Virio harveyii. fragment in the Artemia serves as a model system to establish conditions for the eventual expression of functional antibody Example 2 fragments that will target surface determinants of different viral and bacterial marine pathogens. Binding affinity and Transgenic Microorganism Expressing Antibody specificity of rDB3 closely resemble those of the parent IgG1 Fragments antibody; the binding constant is in the order of 1X10 litres/ mol. Expression and secretion of rDB3 was under control of 0146 Lines of marine cyanobacteria, algae and diatoms— a heterologous promoterisignal peptide complex derived common components of feed for farmed shrimp and fish— from the alpha antigen gene (MKoy) of Mycobacterium kan were transformed to produce antibodies that neutralize infec sasii (Matsuo et al. 1990). The R. rhodnii replication origin tious pathogens such as WSSV and Vibrio. Delivery of these fragment from the shuttle plasmid pR1.1 (Beard et al., 1992) feed organisms, either in slurry preparations or via a bioam was restricted and cloned into the EcoR1 restriction site of the plification strategy with Artemia, results in passive immuni DNA vectorpBluescript SK-- (Stratagene). MKoy was ampli Zation of the alimentary tract of farmed marine animals. fied using the polymerase chain reaction (PCR) oligonucle 0147 This is the portal of entry for many infectious agents otide primers: (SEQID NO: 1) 5'-GCTCT AGA GTT AAC and the delivery of neutralizing antibodies would either abort TATTCTTTG TAC GCG-3 (forward) and (SEQID NO: 2) the infectious process or delay it sufficiently to permit har 5'-GC GAA CGCTCC CGC GGT CGC-3'(reverse). The vest. We have demonstrated that a marine cyanobacterium, forward primer incorporated a 5' Xbal site and the reverse Synechococcus bacillarus, was genetically transformed to primer contained a native Sac II site. The gene encoding the express a functional recombinant antibody (Durvasula et al. single-chain antibody fragment DB3VH/K was amplified 2006, incorporated herein by reference in its entirety). S. using the PCR oligonucleotide primers (SEQ ID NO: 3): bacillarus was transformed to produce a murine antibody 5'-GCACC GCG GGAGCC CAG GTG AAA CTG CTC-3' (rDB3) against progesterone, using a heterologous expres (forward) and (SEQID NO:4): 5'-CCTCGATTGCGGCCG sion system. In competitive ELISA studies, the rDB3 anti CTTAAC-3' (reverse). The forward primer included a Sac II body bound progesterone in a dose-dependent and specific site which allowed for ligation in frame with the DB3 frag manner. No cross-reactivity with testosterone, a structurally ment and the MKa sequence. The reverse fragment contained similar steroid, was detected. This study demonstrated that a a 3' Xbal site. The ligated MKaDB3VH/K fragment was US 2008/0107652 A1 May 8, 2008

cloned into the ball site of the shuttle vector. Cloning of a inhibitor at concentrations that would yield 50% of the maxi kanamycin resistance gene (Pharmacia) as a Bam HI frag mal binding as detected by odometer readings. The mixture ment yielded the final shuttle plasmid pRrMDWK6. Trans contained free progesterone-3-carboxymethyloxime (CMO) formation of S. bacillarus with pRrMDWK6 was done as and was incubated on progesterone-BSA coated plates. The previously described (Durvasula et al 2006, incorporated binding was detected as in the ELISA assay above. herein by reference in its entirety). Example 7 Example 5 Establishment of Paratransgenic Artemia spp. Detection of MDWK6 Shuttle Plasmid in 0153. One to two (1-2) Liter of transformed Synechococ Synechococcus sp. cus was cultured in Seawater-LB broth containing 50 lug/ml 0150 Individual colonies of the transformed Synechococ kanamycin. After 2-3 days of growth, it was centrifuged at cus were picked and were grown in one liter of F/2 with 20% 5000 rpm for 15 m and the media was drained off. The cells BHI, G medium additions and kanamycin (501 g/ml). A mini were Suspended in sterilized normal saline and centrifuged mum inhibitory concentration (MIC) was completed by for another 15 min at 4000 rpm. This step repeated for 4 times growing transformed and wild-type Synechococcus in to remove the residual media and kanamycin. The final cells increasing concentrations ofkanamycin (25, 50, 75, 100, 200, were suspended in 10 ml (2X10) normal saline. - 250 ug kan/ml). The transformed Synechococcus had a 0154 Artemia eggs (Brine Shrimp Direct Inc) were MIC of 250 ug/ml in comparison with the non transformed allowed to hatch in sterilized sea water. Three days after Synechococcus which had a MIC of <50 ug/ml. hatching, the hatchlings were transferred to 20 L. glass aquarium with properaeration. One batch of Artemia was fed 0151 PCR was performed on the Synechococcus lysate with transformed Synechococcus spp. (2X107 cells/ml) and using primers specific to the kanamycin resistance gene. another fed with untransformed Synechococcus (2X107 KANF 5' (SEQ ID NO. 5): GCTCAGTGGAAC cells/ml). Feeding was repeated once in 2 days for 6 days. A GAAAACTCA and KANR5': (SEQ ID NO: 6) CAATTA known Volume of Artemia were harvested once in 2 days and CAAACAGGAATCGAATG. 5ul of lysate was used as tem were thoroughly washed 8-10 times with sterilized seawater plate. The PCR was performed under the following cycling and filtered through a 0.4 micrometer to remove any cynao conditions: 1) a single cycle of 90° C. for 3 minutes followed baceteria adhered on the surface. This Artemia sample was by 30 cycles of 94° C. for 1 minute, 52° C. for 1 minute, 72° used for further molecular analysis. Expression and secretion C. for 30 seconds and a single cycle elongation step of 72°C. of a functional mouse specific progesterone binding antibody for 10 minutes. The kanamycin resistant fragment of 500 bp from paratransgenic Artemia was measured by plating a por could be amplified only from the transformed Synechococ tion of Sonicated Artemia lysate in seawater agar containing CS. 50 ul/mlkanamycinto detect the growth of any colonies in the Example 6 plates. Alternatively we transformed Synechococcus bacilla rus with a plasmid CD3-377 (ABRC, Columbus, Ohio) Expression and Secretion of a Functional Mouse expressing GFP protein. Fluorescence microscopy revealed Specific Progesterone Binding Antibody that protein expressed by cyanobacteria was present in the gut of paratransgenic Artemia fed with S. bacillarus expressing 0152 Western blot analysis was performed on the untrans GFP. formed and transformed Synechococcus lysate. An SDS PAGE gel was run with 100 g of total protein content. The Example 8 blot was then transferred to a PVDF membrane (Immobilon, Milipore) and blocked with 5% non-fat milk in TBS (Tris Expression of Peneidin-Like Antimicrobial Peptide buffered saline containing 1% tween 20) and washed thor (AMP) in Paratransgenic Artemia spp. oughly with TBS. The blot was then probed with an HRP 0.155 Penaeidin-like AMP was cloned and characterized linked secondary anti-mouse antibody at a dilution of 1:5000 from the hemocytes of Tiger shrimp (Penaeus monodon). The and developed using an ECL chemiluminescent detection deduced amino acid sequence of this antimicrobial peptide system (Perkin Elmer). Reactive bands were detected using consisted of 55 amino acid residues of the mature peptide and BioMax MR film (Eastman Kodak, Rochester, N.Y., USA) a signal peptide of 19 amino acids with potent antibacterial only in the lanes that had the transformed Synechococcus activity against Vibrio harveyi, Vibrio alginolyticus and Aero lysate showing that a mouse specific antibody can be coccus viridans (Chiou et al. 2005). We have made de novo expressed by the transformed Synechococcus. ELISA and synthesized gene for AMP by adjusting the codon bias for competitive ELISA were performed on the transformed and optimal cyanobacterial protein expression according to Wil untransformed S lysate. Progesterone and testosterone at a beret al. 1990, incorporated herein by reference inits. A SacII concentration of 3 ug/well were coated on microtiter plates. and Xbal restriction site was inserted at the 5' and 3' end Lysates from transformed and nontransformed Synechococ respectively in the gene sequence to clone into our shuttle cus that were diluted serially were added to the progesterone plasmid, pRrMDWK6. (BSA conjugate, Sigma) or testosterone (BSA conjugate, Fitzgerald) coated wells and incubated at 16° C. overnight. Example 9 The secondary antibody used was AP-linked anti-mouse IgG (Chemicon). Color was developed by addition of 4-Nitrophe Bacterial Challenge Studies with Vibrio Harveyi in nol Phosphate tablets (Roche) and read at 410 nm. A com Shrimp Fed Paratransgenic Artemia Spp. petitive ELISA using free progesterone was also conducted 0156 Two routes of feeding of P. monodon nauplii was similar to He, et al. Here, the progesterone was used as an used in these studies. In one set of studies, transformed cyano US 2008/0107652 A1 May 8, 2008 bacteria was fed directly via a wet feed preparation. The other 0166 Biering, E. S. Villoing, I Sommerset, and K E route was involved feeding Artemia that have accumulated Christie. 2005. Update on viral vaccines for fish. Dev Biol transgenic cyanobacteria. In either case, we assessed gut (Basel) 121:97-113. expression levels of the recombinant sclv or peptide. P. mon Odon nauplii was challenged with Vibrio harveyi as per the 0.167 Boudinot, P., M. Blanco, P. de Kinkelin, and A. protocols of Chen et al. (2000), incorporated herein by refer Benmansour. 1998. Combined DNA immunization with ence in its entirety. We measured total mortality in the experi the glycoprotein gene of viral hemorrhagic septicemia mental group (carrying genetically transformed Synechococ virus and infectious hematopoietic necrosis virus induces cus) versus the control groups (carrying wild-type double-specific protective immunity and nonspecific Synechococcus or Synechococcus expressing an inert marker response in rainbow trout. Virology 249:297-306. antibody reference). Each trial involved 1000 P. monodon 0168 Boynton, J. E., N. W. Gillham, E. H. Harris, J. P. nauplii and performed in triplicate. In Summary, we tested Hosler, A.M. Johnson, A. R. Jones, B. L. Randolph-Ander pathogen specific molecules via 2 different feed strategies in son, D. Robertson, T. M. Klein, K.-B. Shark, and et al. this study. The protection against V harveyi was calculated as 1988. Chloroplast transformation in Chlamydomonas with the relative percent survival. Statistical analysis of the sur high velocity microprojectiles. Science 240: 1534-8. vival rates among the groups was performed using the chi square test at a 5% confidence level. The results demonstrated 0169 Brown, L. E., S. L. Sprecher, and L. R. Keller. 1991. full mortality in the control groups and statistically significant Introduction of exogenous DNA into Chlamydomonas increase in Survival in the groups fed transgenic cyanobacte reinhardtii by electroporation. Mol Cell Biol 11:2328-32. 18. Campbell, R. A. Adams, M. F. Tatner, M. Chair, and P. 0157 All references discussed herein are specifically Sorgeloos. 1993. Uptake of Vibrio angillarum vaccine by incorporated herein by reference. One skilled in the art will Artemia salina as a potential oral delivery system to fish readily appreciate that the present invention is well adapted to fry. Fish Shellfish Immunnol 3:451–459. carry out the objects and obtain the ends and advantages 0170 Chair, M., M. Dehasque, S. Van Poucke, H. Nelis, P. mentioned, as well as those inherent therein. 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0205 Kanellos, T.I. D. Sylvester, C. R. Howard, and P. H. 0219 Rabbani, S. P. Beyer, J. Lintig, P. Hugueney, and H. Russell. 1999. DNA is as effective as protein at inducing Kleinig. 1998. Induced beta-carotene synthesis driven by antibody in fish. Vaccine 17:965-72. triacylglycerol deposition in the unicellular alga 0206 Kim, C. H., M. C. Johnson, J. D. Drennan, B. E. Dunaliella bardawil. Plant Physiol 116:1239-48. Simon, E. Thomann, and J. A. Leong. 2000. DNA vaccines 0220 Raengpipat, S. W Phianphak, S Piyatiratitivorakul, encoding viral glycoproteins induce nonspecific immunity and P Menasveta. 1998. Effects of probiotic bacterium in and Mx protein synthesis in fish. JVirol 74:7048-54. black tiger shrimp Penaeus monodon Survival and growth. 0207 Kurath, G. K. A. Garver, S. Corbeil, D. G. Elliott, E. Aquaculture 167:301-313. D. Anderson, and S. E. LaPatra. 2006. Protective immunity 0221 Rahman, A., and N. Maclean. 1992. 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SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 6

<210 SEQ ID NO 1 <211 LENGTH: 29 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 1

gctictagagt taact attct ttgtacgcg 29

<210 SEQ ID NO 2 <211 LENGTH: 2O &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 2 gcgaacgct C cc.gcggit cqc

<210 SEQ ID NO 3 <211 LENGTH: 29 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 3 US 2008/0107652 A1 May 8, 2008 19

- Continued gCaccgcggg agcc.caggtgaaactgctic 29

SEQ ID NO 4 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer

<4 OO SEQUENCE: 4

Ccticgattgc ggcc.gcttaa C 21

SEO ID NO 5 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer

<4 OO SEQUENCE: 5 gct cagtgga acgaaaactic a 21

SEQ ID NO 6 LENGTH: 23 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer

<4 OO SEQUENCE: 6 caattacaaa caggaatcga atg 23

What is claimed is: 8. The paratransgeneic method of claim 1, wherein the 1. A paratransgenic method for prevention, amelioration or recombinant molecule comprises one or more antibacterial treatment of a disease or disorder in an aquatic animal com molecules. prising: i) providing a genetically modified microalgae that 9. The paratransgeneic method of claim 8, wherein the expresses one or more recombinant molecules that specifi antibacterial molecules comprise Peneidin-Like antimicro cally target one or more key epitopes of a pathogen that bial peptide AMP. infects the aquatic animal and ii) feeding the aquatic animal 10. The paratransgeneic method of claim 1, wherein the directly or indirectly with the genetically modified micro genetically modified microalgae is bioamplied in a probiotic algae. organism prior to consumption by the aquatic animal. 2. The paratransgeneic method of claim 1, wherein the 11. The paratransgeneic method of claim 3, wherein the microalgae comprises Dunaliella or a variant thereof. probiotic organism comprises bacterium, and planktonic 3. The paratransgeneic method of claim 1, wherein the organism comprising Artemia, rotifers, copepods, or daphnia, recombinant molecule comprises one or more immunogenic or a combination thereof. peptides, single chain antibody fragments, DNA vaccine, or a 12. The paratransgeneic method of claim 1, wherein the combination thereof. genetically modified micro algae is produced by transforma 4. The paratransgeneic method of claim 3, wherein the tion of a symbiotic or commensal bacteria of the microalgae single chain antibody fragment specifically binds to one or with a desired genetic material. more key epitopes of a pathogen. 13. The paratransgeneic method of claim 1, wherein the microalgae comprises a unicellular micro algae. 5. The paratransgeneic method of claim 4, wherein the 14. The paratransgeneic method of claim 13, wherein the pathogen comprises a virus, bacterium, protozoa, or a com microalgae comprises Isochrysis, Pavlova, Nannochloropsis, bination thereof. Thalassiosira psu.edoniana, Cyanobacterium, Dunaliella, 6. The paratransgeneic method of claim 5, wherein the Phaeodactylum tricornutum, Red alga Porphydium cruen single chain antibody fragment blocks assembly of the virus tum, Haematococcus, Botryococcus, Gymnodinium sp., Gon by inhibiting expression of one or more viral proteins. vaulax, Chlamydomonas, Chlorella pyrenoidosa, or species 7. The paratransgeneic method of claim 5, wherein the and variants thereof. virus comprises White Spot Syndrome Virus (WSSV), or 15. The paratransgeneic method of claim 14, wherein the variants and serotypes thereof. cyanobacterium comprises Cyanobacterium Spirulina, US 2008/0107652 A1 May 8, 2008 20

Cyanobacteria Scytoinema, cyanobacteria Oscillatoria, or 19. The paratransgeneic method of claim 1, wherein the Synechococcus bacillarus, or species and variants thereof. antibody fragment is a scFV fragment that provides immunity 16. The paratransgeneic method of claim 1, wherein the against infections by Vibrio harveyi, White Spot Syndrome Virus, or both. aquatic animal is a farm-raised animal. 20. A paratransgeneic method for control of infection in 17. The paratransgeneic method of claim 16, wherein the aquaculture comprising: i) providing a genetically modified aquatic animal comprises shrimp. cynobacteria that expresses a recombinant molecule that spe 18. The paratransgeneic method of claim 1, wherein the cifically targets one or more key peptides of a pathogen that genetically modified micro algae expresses a protein, a pep infects an aquatic animal and ii) feeding the aquatic animal tide, or one or more antibody fragments that inhibit the directly or indirectly with the genetically modified cynobac growth or replication of a pathogen comprising Vibrio spe teria. cies, Taura, and White spot virus.